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CA 02528853 2005-12-23
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CA 02528853 2005-12-23
METHODS AND NUCLEIC ACIDS FOR ANALYSES OF
COLORECTAL CELL PROLIFERATIVE DISORDERS
FIELD OF THE iNVENTION
The present invention relates to genomic DNA sequences that exlubit altered
CpG
mathylation patterns in disease states relative to normal. Particular
embodirnents provide
methods, nucleic acids, nucleic acid arrays and kits useful far detecting, or
for detecting and
differentiating between or among colorectal cell proliferative disorders.
BACKGROUND
The etiology of pathogenic states is kaown to involve modified methylation
patterns of
individual genos or of the genome. 5-methylcytosine, in the context of CpG
dinucleotide
sequences, is the most frequent covalently modified base in the DNA of
eukaryotic cells, and
plays a role in the regnlaRion of truscription, gmtic imprinting, and
tumorigenesis. The
identification and quantification of 5-methyleytosine sites in a specific
specimen, or between or
among a plurality of specimens, is thus of considerable interest, not only in
research, but
particularly for the molocular diagnoses of various diseases.
Carrelatiort of aberrant DNA rnethylation with cancer. Aberrrant DNA
methylation
within CpG 'islands' is charactezized by hyper- or hypomethylation of CpG
dinucleotide
sequences leading to abrogation or overexpression of a broad spacttum of
genes, and is among
the earliest and most common a lterations found in, and correlated with human
malignancies.
t
CA 02528853 2005-12-23
Additionally, abnormal methylation has been shown to occur in CpG-rich
regulatory elements in
intronic and coding parts of genes for certain tumors. In colon cancer, for
example, aberrant
DNA methylation constitutes one of the most prominent alterations and
inactivates many tumor
suppressor genes such as p14ARF, p16TMC4a, THBSI, M1NT2, and 1VIINT3i and DNA
mismatch repair genes such as hMLHI.
In contrast to the specific hypennethylation of tumor suppressor genes, an
overall
hypomethylation of DNA can be observed in tumor cells. This decrease in global
methylation
can be detected early, far before the development of frank tumor formation. A
correlation
between hypomethylation and increased gene expression has been determined for
many
oncogenes.
In the United States the annual incidence of colorectal cancer is
approximately 150,000,
with 56,600 individuals dying form colorectal cancer each year. The lifetime
risk of colorectal
cancer in the general population is about 5 to 6 percent. Despite intensive
efforts in recent years
in screening and e arly d etection o f c olon cancer, until today most cases
are diagnosed in an
advanced stage with regional or distant metastasis. W'hile the therapeutic
options include
surgery and adjuvant or palliative chemotherapy, most patients die from
progression of their..
cancer within a few months. Identifjring the molecular changes that underlie
the development of
colon cancer may help to develop new monitoring, screening, diagnostic and
therapeutic options
that could improve the overall poor prognosis of these patients.
The current guidelines for colorectal screening according to the American
Cancer
Society utilizes one of five different options for screening in average risk
individuals 50 years of
age or older. These options include 1) fecal occult blood test (FOBT)
annuaily, 2) flexible
sigmoidoscopy every five years, 3) annual FPBT plus flexible signioidoscopy
every five years,
4) double contrast barium enema (DCBE) every five years or 5) colonoscopy
every ten years.
Even though these testing procedures are well accepted by the medical
community, the
implementation of widespread screening for colorectal cancer has not been
realized. Patient
compliance is a major factor for limited use due to the discomfort or
inconvenience associated
with the procedures. FOBT testing, although a non-invasive procedure, requires
dietary and
other restrictions 3-S days prior to testing. Sensitivity levels for this test
are also very low for
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CA 02528853 2005-12-23
coloroctal adenocarcinoma with wide variability depending on the trial.
Sensitivity
measurements for detection of adenomas is even less since most adenomas do not
bleed. In
contrast, sensitivity for more invasive procedures such as sigmoidoscoapy and
colonoscopy are
quite high because of direct visualization of the hunen of the colon. No
randomized trials have
evaluated the efficacy of these techniques, however, using data from case-
control studies and
data from the National Polyp Study (U.S.) it has been shown that removal of
adenomatous
polyps results in a 76-90% reduction in CRC incidence. Sigmoidoscopy has the
lianitation of
only visualizing the left side of the colon leaving lesions in the right colon
undetected. Both
scoping procedures are expensive, require cathartic preparation and have
increased risk of
morbidity and mortality. Improved tests with increased sensitivity,
specificity, ease of use and
decreased costs are clearly needed before general widespread screening for
colorectal cancer
becomes routine.
Early colorectal cancer detection is generally based on the fecal occult blood
test (FOBT)
performed annually on asymptomatic individuals. Cuzent recommendations adapted
by several
healthcare organizations, including the American Cancer Society, call for
fecal occult blood
testing beginning at age 50, repeated annually until such time as the patient
would no longer
benefit from scroening. A positive FOBT leads to colonoscopic exanaination of
the bowel; an
expensive and invasive procedure, with a serious complication rate of one per
5,000
examinations. Only 12% of patients with home positive stool are diagmosed with
cancer or large
polyps at the time of colon~oscopy. A number of studies show that FOBT
screening does not
improve cancer-related mortality or overall survivat. Compliance with occult
blood testing has
been poor; less than 20 percent of the population is offered or completes FOBT
as
recommanded. If FOBT is properly done, the patient collects a fecal sample
from three
consecutive bowel movements. Samples are obtained while the patient adheres to
dietary
guidelines and avoids medications known to induee occult gastrointestinal
bleeding. In reality,
physicians frequently fail to instruct patients properly, patients frequently
fail to adhere to
pmtocol, and some patients find the task of collecting fecal samples difficult
or unpleasant,
hence compliance with annual occult blood testing is poor. If testing
sensitivity and specificity
can be improved over current methods, the frequency of testing could be
reduced, eollection of
3
CA 02528853 2005-12-23
consecutive samples would be eliminated, dietary and medication schedule
modifications would
be eliminated, and patient compliance would be enhanced. Compounding the
problem of
compliance, the sensitivity and specificity of FOBT to detect colon cancer is
poor. Poor test
specificity leads to unnecessary colonoscopy, adding considerable expense to
colon cancer
screening.
Specificity of the FOBT has been calculated at best to be 96 /., with a
sensitivity of 43%
(adenomas) and 50% (colorectal carcinoma). Sensitivity can be improved using
an
immunoassay FOBT such as that produced under the tradcname 'InSure"m', with an
improved
sensitivity of 77 % (adenomas) and 88.9~0 (colorectal carcinoma.
Molecular disease markers offer several advantages over other types of
maYkers, one
advantage being that even samples of very small sizes and/or samples whose
tissue architecture
has not been maintained can be analyzed quite efficiently. Within the last
decade a number of
genes have been shown to be differentially expressed between normal and colon
carcinomas.
However, no siagle or combination of marker has been shown to be su.fficient
for the diagnaais
of colon carcinomas. High-dimensional'mRNA based approaches have recently been
shown to
be able to provide a better means to distinguish between different tumor types
and benign and
naalignant lesions. However its application as a routine diagnostic tool in a
clinical environment
is impeded by the extrame instability of mRNA, the rapidly oocurting
expression changes
following certain triggers (e.g., sample collection), and, most importantly,
the large amount of
mRNA needed for analysis (Lipshutz, R. J. et al., Nature Genetics 21:20-24,
1999; Bowtell, D.
D. L. Nature genetics suppl. 21:25-32, 1999), which often cannot be obtained
from a routine
biopsy.
The use of biological markers to further improve sensitivity and specificity
of FOBT has
been suggested, examples of such tests include the PreGen-PlusTM stool
analysis assay available
from EXACT Sciences which has a sensitivity of 20% (adenoma) and 52%
(colorectal
carcinoma) and a specificity of 95% in both cases. This test assays for the
presence of 23 DNA
mutations associated with the development of colon neoplasms. The use of DNA
methylation as
colon cancer markers is known. For example Sabbioni et al. (Molecular
Diagnosis 7:201-207,
2003) detected hypermethylation of a panel of gc,nes consisiting TPEF, HICI,
DAPK and
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CA 02528853 2005-12-23
MGMT in peripheml blood in 98% of colon carcinoma patients. However, this does
provide a
suitable basis for a commercially marketable test, as the specificity of such
a test must also be
sufficiently high.
The cunreqtmodel of colorectal pathogenesis favours a stepwise progression of
adenomas, which includes the development of dysplasia and finally sigas of
invasive cancer.
The molecular changes underlying this adenoma-careinoma sequence include
genetic and
epigenetic alterations of tumor suppressor genes (APC, p53, DCC), the
activation of oncogenes
(K-ras) and the inactivation of DNA mismatch repair genes. Recently, further
molecular
changes and genetic defects have been revealed. Thus, activation of the Wnt
signalling pathway
not o nly includes m utations o f the A PC gene, b ut m ay also r esult from P
-catenin m utations.
Furthermore, a lterations i n t he T GF-0 s ignalling p athway t ogether w ith
its s ignal t ransd.ucers
SMAD4 and SMAD2 have been linked to the development of colon cancer.
Despite recent progress in the understanding of the pathogenesis of adenomas
and
carcinomas o f t he c=olon a nd t heir genetic a nd molecular changes, t he
genetic and e pigenetic ;
changes underlying the development of metastasis are less well understood. It
is, however,
generally well accepted that the process of invasion and proteolysis of the
extracellular matrix; :
as well a s infiltration of the vascular basement m embrane involve adhesive
proteins, such as,
members of the family of integrin receptors, the cadherins, the immunoglobulin
superfamily, the
laminin binding pratein and the CD44 receptor. Apart from adhesion, the
process of metastasis
formation also includes the induction and regulation of angiogenesis (VEGF,
bFGF), the
induction of cell proliferation (EGF, HGF, IGF) and the aativation of
proteolytic enzymes
(AWs, TIMPs, uPAR), as well as the inhibition of apoptosis (Bcl-2, Bcl-X).
More recently
other groups have compared the genetic and molecular changes in metastatic
lesions to the
changes found in primary colorectal cancers. Thus, Kleeff et al. reported the
loss of DOC-2, a
candidate tumor suppressor gene, both in primary and metastatic colorectal
cancer.
Furthermore, Zauber et al. reported that in their series of 42 colorectal
cancers Ki-ras mutations
in the primary cancers were identical in all of the 42 paired primary and
synchronous metastatic
lesions. Similarly loss of heterozygosity at the APC locus was identical for
39 paired
carcinomas and synchronous metastasis. The authors concluded that for Ki-ras
and APC genes
5
CA 02528853 2005-12-23
the genetic changes in metastasis are identical to the primary colorectal
cancer. However, other
groups have found genetic and molecular changes in metastatic colon cancers,
that are not
present in the primary cancers. Thus, the development of LOH of chromosome 3p
in colorectal
metastasis has been reported. In addition, using comparative genomic
hybridization several
alterations were found in liver metastasis that were unique to metastastic
lesions (-9q, -11q, and
-17q).
Apart from mutations aberrant methylation of CpG islands has been shown to
lead to the
transcriptional silencing of certain genes that have been previously linked to
the pathogenesis of
various cancers. CpG islands are short sequences which are rich in CpG
dinucleotides and can
usually be found in the 5' region of approximately 50% of all human genes.
Methylation of the
cytosines in these islands leads to the loss of gene expression and has been
reported in the
inactivation of the X cbromosome and genomic imprinting.
Recently several groups have also analysed the methylation of various genes in
colorectal cancer and reported the transeriptional silencing by promoter
methylation for
p16INK4, p14ARF, pl5IlVK4b, MGMT, hMLH1, GSTPI, DAPK, CDHI, T3Tvl.P-3 and APC
among others. Thus apart from mutational inactivation of certain genes, the
hyperttxethylation of,=
these genes also contributes significantly to the pathogenesis of this
disease.
In recent years several genes that are methylated in colon cancer have been
identified by
MS-APPCR. This group of genes, among others, includes TPEF/HI'P1 which is
frequently
methylated i n c olon c ancers a nd w hich w as i ndependently i dentified b y
t wo d ifferent g roups
using the MS-APPCR method (see, e.g., Young J, Biden KG, Simms LA, Huggard P,
Karamatic
R, Eyre HJ, Sutherland GR, Herath N, Barker M, Anderson GJ, Fitzpatrick DR,
Ramm GA, Jass
JR, Leggett BA. HPP1: a transmembrane protein-encoding gene commonly
methylated in
colorectal polyps and cancers. Proc Natl Acad Sci USA 98:265-270, 2001).
Multifactorfal approach. Cancer diagnostics has traditionally relied upon the
detection of
single molecular markers (e.g., gene mutations, elevated PSA levels).
Unfortunately, cancer is a
disease state in which single markers have typically failed to detect or
differentiate many forms
of the disease. Thus, assays that recognize only a single marker have been
shown to be of
limited predictive value. A fundamental aspect of this invention is that
methylation-based
6
CA 02528853 2005-12-23
cancer diagnostics and the screening, diagnosis, and therapeutic monitoring of
such diseases will
provide significant improvements over the stat"f-the-art that uses single
marker analyses by
the use of a selection of multiple markers. The multiplexed analytical
approach is particularly
well suited for cancer diagnostics since cancer is not a simple disease, this
multi-factorial
"panel" approach is consistent with the heterogeneous nature of cancer, both
cytologically and
clinically.
Key to the successful implementation of a panel approach to methylation based
diagnostic tests is the design and development of optimized panels of markers
that can
characterize and distinguish disease states.
Development of medical tests. Two key evaluative measures of any medical
screening or
diagnostic test are its sensitivity and specificity, which measure how well
the test performs to
accurately detect all affected individuals without exception, and without
falsely including
individuals who do not have the target disease (predicitive value).
Historically, many diagnostic
tests have been criticized due to poor sensitivity and specificity.
A true positive (TP) result is where the test is positive and the condition is
present. A
false positive (FP) result is where the test is positive but the condition is
not present. A true,
negative (TN) result is where the test is negative and the condition is not
present. A false !
negative (F'N) result is where the test is negative but the condition is not
present. In this context:
Sensitivity = TP/(TP+FN); Specificity = TN/(FP+TN); and Predictive value =
TP/(TP+FP).
Sensitivity is a measure of a test's ability to correctly detect the target
disease in an
individual being tested. A test having poor sensitivity produces a high rate
of false negatives,
i.e., individuals who have the disease but are falsely identified as being
free of that particular
disease. The potential danger of a false negative is that the diseased
individual will remain
undiagnosed and untreated for some period of time, during which the disease
may progress to a
later stage wherein treatments, if any, may be less effective. An example of a
test that has low
sensitivity is a protein-based blood test for HN. This type of test exhibits
poor sensitivity
because it fails to detect the presence of the virus until the disease is well
established and the
virus has invaded the bloodstream in substantial numbers. In contrast, an
example of a test that
has h igh s ensitivity i s v iral-load d etection u sing t he p olymerase c
hain r eaction (PCR). H igh
7
CA 02528853 2005-12-23
sensitivity is a chieved b ecause this type o f t est can detect very s mall
quantities o f the virus.
High sensitivity is particularly important when the consequences of missing a
diagnosis are
high.
Specificity, on the other hand, is a measure of a test's ability to identify
accurately
patients who are free of the disease state. A test having poor specificity
produces a high rate of
false positives, i.e., individuals who are falsely identified as having the
diseasa A drawback of
false positives is that they force patients to undergo unnecessary medicat
procedures treatments
with their attendant risks, emot-onal and financial stresses, and which could
have adverse effects
on the patient's health. A feature of diseases which makes it difficult to
develop diagnostic tests
with high specificity is that disease mechaaisms, particularly in cancer,
often involve a plurality
of gam and proteins. Additionally, certain proteins may be elevated for
reasons unrelated to a
disease state. n example of a test that has high specificity is a gene-based
test that can detect a
p53 mutation. Specificity is important when the cost or risk associated with
fiuther diagnostic
procednres or farther medieal intervention are very high.
Pronounced need in the art. If colon cancer screening specificity can be
increased, the
problem of false positive test results leading to unnecessary colonoscopic
examination would be
reduced leading to cost savings and impmved safety.
In view of the incidence of colon cancer andd disadvantages associated with
cumant
colorectal cell proliferative disorder screening methods there is a
substantial need in the art for
ixnproved methods for the early detection of colon cell proliferative
disorders, in particular colon
cancer, to be used in addition to or as a substitute for currently available
tests.
SUMMARY OF THE YNVENTION
Various aspects of the present invention provide efficient and unique panels
of genes,
whereby methylation analysis of one or a combination of the members of the
panel enables the
detection of cell proliferative disorders of the prostate with a particularly
high sensitivity,
specificity and/or predictive value. The inventive colorectal cancer testing
methods have
particular utility for the screening of at-risk populations. The inventive
methods have
advantages over prior art methods (including the industry standard FOBT),
because of improved
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CA 02528853 2005-12-23
sensitivity, specificity and likely patient compliance.
The present invention provides novel metlto(Is for detecting or distinguisWng
between
colorectal cell prolifera.tive disorders. Said methods are most preferably
utilised for detecting or
detecting and distinguishing between one or more of the following: colorectal
carcinoma, colon
adenoma, inflammatory colon tissue, grade 2 dysplasia colon adenomas less than
1 cm, grade 3
dysplasia colon adenomas larger than 1 cm, normal colon tissue, non-colon
normal tissue, body
fluids and non-colon cancer tissue.
In a farther embodiment of the invention the method according to the invention
may be
used for the detection of aerodigestive cell proliferative disorders.
In one embodiment the invention provides a method for detecting and/or for
detecting
and distinguishing between or among colorectal cell proliferative disorders in
a subject. Said
method comprises the following steps: i) contacting genomic DNA isolated from
blood plasma,
blood serum, whole blood, isolated blood cells, cells isolated from the blood
obtained from the
subject with at least one reagent, or series of reagents that distinguishes
between methylated and
non-methylated CpG dinucleotides within at least one target region of the
genomic DNA,'
wherein the nucleotide sequence of said target region comprises at least one
CpG dinucleotide
sequence; and ii) detecting, or detecting and distingnishing between or among
colorectal cell ,
proliferative disorders afforded with a sensitivity of greater than or equal
to 80% and a
specificity of greater than or equal to 80%.
Preferably, the sensitivity is from about 75% to about 96%, or from about 80%
to about
90o/a, or from about 80% to about 85%. Perferably, the specificity is is from
about 75% to about
96%, or from about 80% to about 90%, or from about 80% to about 85%.
The method is novel as no methods currently exist that enable the detection o
f c olon
cancer by analysis of bodily fluids, with a sensitivity and specificity high
enough for use in a
commercially available and regulatory body approved assay. Current methods
used to detect
and diagnose colorectal cell proliferative disorders include colonoscopy,
sigmoidoscopy, and
fecal occult blood colon cancer. In comparison to these methods, the disclosed
invention is
much less invasive than colonoscopy, and as, if not more sensitive than
sigmoidoscopy and
FOBT. The development of a body fluid assay represents a clear technical
advantage over
9
CA 02528853 2005-12-23
current methods known in the art in that it is anticipated that patient
compliance for a single
body fluid based test will be higher than the triplicate analysis of stool
currently recommended
for FOBT.
A particular embodiment the method comprises the use of one or more genes or
genomic
sequences selected from the group consisting of: SEQ ID NOS:1-3, 14, 16, 21,
22, 23, 44-46,
48, 50, 59-64, COX7B, CST5, EYA3, FTH1, RNF4, SOX 21, RNF4, ALX 4, ONC2, NGFR,
DLX-5, HVIAAT, BTF3 Homolog 1, ADCY9, BCL-5, BCOR, TAFU28, HOMEOBOX
PROTEIN SIX6, GSK-3 BETA, ELK-L, HLA-F, TGFBR2, AR, RNF30 and DUX4 as markers
for the differentiation, detection and distinguishing of colorectal cell
proliferative disorders.
Said use of the gene may be enabled by means of any anal.ysis of the
expression of the
gene, by means of mRNA expression analysis or protein expression analysis.
However, in the
most preferred embodiment of the invention, the detection, differentiation and
distinguishing of
colorectal cell proliferative disorders is enabled by means of analysis of the
methylation status
of one or more genes or genomic sequences selected from the group consisting
of SEQ ID
NOS:1-3, 14, 16, 21, 22, 23, 44-46, 48, 50, 59-64, COX7B, CST5, EYA3, FTHI,
RNF4, SOX
21, RNF4, ALX 4, ONC2, NGFR, DLX-5, HVIA.AT, BTF3 Homolog 1, ADCY9, BCIrSõ
BCOR, TAFII28, HOMEOBOX PROTEIN SIX6, GSK-3 BETA, ELK-L, HLA-F, TGFBR2, ..
AR, RNF30, DUX4, and their promoter or regulatory elements.
The invention provides a method for the analysis of biological samples for
features
associated with the development of colorectal ceil proliferative disorders,
the method
characterised in that at least one nucleic acid, or a fragment thereof, from
the group consisting of
SEQ ID NO:1 to SEQ ID NO:535 is/are contacted with a reagent or series of
reagents capable of
distinguishing between methylated and non methylated CpG dinucleotides within
the genomic
sequence, or sequences of interest.
The present invention provides a method for ascertaining genedc and/or
epigenetic
parameters of genomic DNA. The method has utility for the improved diagnosis,
treatment and
monitoring of colorectal cell proliferative disorders, more specifically by
enabling the improved
identification of and differentiation between subclasses of said disorder. The
invention presents
improvements over the state of the art in that it enables a highly specific
classification of
CA 02528853 2005-12-23
colorectal celt proliferative disordeis, thereby allowing for improved and
informed trcatment of
patients.
Preferably, the source of the test sample is selected from the group
consisting of cells or
cell lines, histological s2ides, biopsies, paraffin-embedded tissue, bodily
fluids, ejaculate, stool,
urine, blood, and combinations thereof. Preferably, the source is selected
from the group
consisting of stool, blood plasma, blood serum, whole blood, isolated blood
cells, cells isolated
from the blood obtained from the subject.
Specifically, the present invention provides a method for detecting colorectal
cell
proliferative disorders, comprising: obtain.ing a biological sample comprising
genoanic nucleic
acid(s); contacting the nucleic acid(s), or a fragment theraof, with one
reagent or a plurality of
reagents sufficient for distinguishing between methylated and non methylated
CpG dinuclefltide
sequences witliin a target sequence of the subject nucleic acid, wherein the
target sequence
comprises, or hybridizes under stringent conditions to, a sequence comprising
at least 16
contigaous nucleotides of SEQ ID NOS:1 to 64, said contiguous nucleotides
camprising at least
one CpG dinucleotide sequence; and determining, based at least in part on said
distinguishing,
the methylation state of at least one target CpG dinucleotide sequence, or an
average, or a value,
reflecting an average methylation state of a plurality of target CpG
dinucleotide sequences.
Preferably, distingaishing between methylated and non methylated CpG
dinucleotide sequences
within the target sequence comprises methylation state-dependent conversion or
non-conversion
of at least one such CpG dinucleotide sequence to the corresponding converted
or non-converted
dinucleotide sequence within a sequence selected from the group consisting of
SEQ ID
NOS:304 ta SEQ ID NO:535 and SEQ LD NOS:65 to SEQ ID NO:88, and contiguous
regions
thereoÃcorresponding to the target sequence.
Additional embodiments provide a method for the detection of colorectal cell
proliferative disorders, comprising: obtaining a biological sample having
subject genomic DNA;
extracting the g enomic DNA; t reating the genomic DNA, o r a fragment t
hereoi; w ith o ne o r
more reagents to convert 5-position unmethylated cytosine bases to uracil or
to another base tbat
is detectably dissimilar to cytosine in terms of hybridization properties;
contacting the treated
genomic DNA, or the treated fragment thereof, with an amplification enzyme and
at least two
I 1
CA 02528853 2005-12-23
primers comprising, in each case a contiguous sequence at least 9 nucleotides
in length that is
complementary to, or hybridizes under moderately stringent or stringent
conditions to a
sequence selected from the group consisting SEQ ID NOS:304 to SEQ ID NO:535
and SEQ ID
NOS:65 to SEQ ID NO:88, and complements thereof, wherein the treated DNA or
the fragment
thereof is either amplified to produce an amplificate, or is not amplified;
and determining, based
on a presence or absence of, or on a property of said amplificate, the
methylation state of at least
one CpG dinucleotide sequence selected from the group consisting of SEQ ID
NOS:1 to SEQ ID
NO:64, or an average, or a value reflecting an average methylation state of a
plurality of CpG
dinucleotide sequences thereof. Preferably, at least one such hybridizing
nucleic acid molecule
or peptide nucleic acid molecule is bound to a solid phase. Preferably,
determining comprises
use of at least one method selected from the group consisting o~ hybridizing
at least one nucleic
acid molecule comprising a contiguous sequence at least 9 nucleotides in
length that is
complementary to, or hybridizes under moderately stringent or stringent
conditions to a
sequence selected from the group consisting of SEQ ID NOS:304 to SEQ ID NO:535
and SEQ
ID NOS:65 to SEQ ID NO:88, and complements thereoF hybridizing at least one
nucleic acid
molecule, bound to a solid phase, comprising a contiguous sequence at least 9
nucleotides in
length that is complementary to, or hybridizes under moderately stringent or
stringent oonditions .
to a sequence selected from the group consisting of SEQ ID NOS:304 to SEQ ID
NO:535 and
SEQ ID NOS:65 to SEQ ID NO:88, and complements thereof; hybridizing at least
one nucleic
acid molecule comprising a contiguous sequence at least 9 nucleotides in
length that is
complementary to, or hybridizes under moderately stringent or stringent
conditions to a
sequence selected from the group consisting of SEQ ID NOS:304 to SEQ ID NO:535
and SEQ
ID NOS:65 to SEQ ID NO:88, and complements thereof, and extending at least one
such
hybridized nucleic acid molecule by at least one nucleotide base; and
sequencing of the
amplificate_
Further embodiments provide a method for the analysis of colorectal cell
proliferative
disorders, comprising: obtaining a biological sample having subject genomic
DNA; extracting
the genomic DNA; contacting the genomic DNA, or a fragment thereof, comprising
one or more
sequences selected from the group consisting of SEQ ID NOS: 1 to SEQ ID NO:64
or a sequence
12
CA 02528853 2005-12-23
that hybridizes under sdringent conditions thereto, with one or more
methylation-sensitive
restriction enzymes, wherain the ge,nomic DNA is either digested thereby to
produce digestion
fragments, or is not digested thereby; and determining, based on a presence or
absence of, or on
property of at least one such fragment, the methylation state of at least one
CpG dinucleotide
sequence of one or more sequences selected fmm the group consisting o f SEQ ID
NOS:1 to
SEQ Ii) N0:64, or an average, or a value reflecting an average methylation
state of a plurality of
CpG dinucleotide sequences thweo~ Preferably, the digested or undigested
genomic DNA is
amplified prior to said determining.
Additional embodiments provide novel genomic and chemieally modified nucleic
aeid
sequences, as well as oligonucleotides and/or PNA-oligomers for analysis of
cytosine
methylation patterns within sequences from the group cons'ssting of SEQ ID
NOS:1 to SEQ ID
NO:64.
BRIEF DESCRIPTION OF THE DRAWINGS
Figures 1, 5, 9, 13, 16, 20, 24, 28 and 32 show ranked matrices of data
obtained
according to EXAMPI.ES 1 and 2, and according to CpG methylation differenaes
between the
two classes of tissues, using a suitable algorithm. The figures are shown in
greyscale, wherein
the most significant CpG positions are at the bottom of the matrix with
significance decreasing
towards the top. Black indicates total methylation at a given CpG position,
white represents no
methylation at the particular position, with degrees of inethylation
represented in grey, from
light (low proportion of inethylation) to dark (high proportion of
Ynethylation). Each row
represents one specific CpG position within a gene and each column shows the
methylation
profile for the different CpGs for one sample. The p-values for the individual
CpG positions are
shown on the right side. The p-values are the probabilities that the observed
distribution
occurred by chance in the data set.
Figures 2, 6, 10, 17, 21, 25, 29 and 33 show the uncorrected multivariate
LogReg test
results for each comparison. Each individual genomic region of interest is
represented as a
point, the dotted line represents the cut off point for the 25% false
discovery rate.
Figures 3, 7, 11, 14, 18, 22, 26, 30 and 34 show nutlced matrices of data,
obtained
13
CA 02528853 2005-12-23
according to EXAMPLES I and 2, of the accuracy of the genewise linear support
vector
machine cross validations between the two classes of tissues, for the best
performing markers.
The figures are shown in greyscale, wherein the most significant CpG positions
are at the
bottom of the matrix with significance decreasing towards the top. Black
indicates total
methylation at a given CpG position, white represents no methylation at the
partacular position,
with degrees of inethylatson rqaresented in grey, from light (low proportion
of inethylation) to
dark (high proportion of inethylatiou.). Each row represents one specific CpG
position within a
gene and each column shows the methylation profile for the diffecent CpGs for
one sample.
Accuracy values for each individual genomic region of interesi are shown on
the right side.
Figures 4, 8, 12, 15, 18, 23, 27, 31 and 35 show the accuzacy of the genewise
linear
support vector machine cross validation for all genomic regions of each
classification. The
accuracy of each genomic region is represented as black squares, the
specificity as unfilled
diamonds, and the sensitivity as unfilled squares. The accuracy as measured on
the X-axis
ftws the fiwtion of correctly classified samples.
Figure 36 shows the level of inethylation determined by different MSP
MethyLigh04
assays and HeavyMethyff MethyLight'74 assays. The Y-axis shows the degree
ofinethylation.
Tumor samples are represented by white points, and normal colon tissue samples
by black .
points. A significantly higher degree of inethylation was observed in tumor
samples than in
healthy tissue samples.
Fignre 37 shows the Receiver Operating Characteristic curve (ROC curve) of the
SEQ
ID NO:35 -MSP-MethylLightm-Assay for adenocarcinomas according to EXAMPI,E 2.
The
AUC for the MSP-MethylLightm-Assay is: 0.94.
Figure 38 shows the Receiver Operating Characteristic curve (ROC curve) of the
SEQ
ID NO:35 -HM-MethylLightm-Assay for Adenocareinoma according to EXAMPLE 3. The
AUC for the HM MethylLightm-Assay is: 0.91.
Figure 39 shows the level of tnethylation determined by a SEQ TD NO:35 -
HeavyMethyff MethyLightTM' assay according to EXAWLE 3, testing an additional
set of
colon samples (25 adenocarcinoma, 33 normals, and 13 adenomas). The Y-axis
shows the
degree of inethylation within the region of the SEQ ID NO:35 gene
investigated.
14
CA 02528853 2005-12-23
Adeuocminoma samq.sles ace represented by white squaras, and normal colon tism
samples by
black diamonds. A significantly higher degree of inethylation was observed in
tumor samples
than in healthy tissae samples. The level of sigriificance as measured using a
t-test was 0.00424.
Figure 40 shows the Receiver Operating Characteristic curve (ROC curve) of the
SEQ
ID NO:35 HM MethylLight"m-Assay for Adenocaccinoma and Adenoma according to
EXAMPLE 3 (additional sets of samples). The area under an ROC curve (AUC) is a
measure
for the accuracy of a diagnostic test. The AUC for the HM-MethylLightTm-Assay
is 0.81.
Figure 41 shows the Receiver Operating Chaiacteristic curve (ROC curve) of the
SEQ
ID NO:35 AM MethylLight7m-Assay fnr Adenocarcinoma only according to LXAMPLE 2
(additional sets of samples). The area under an ROC curve (AUC) is a measure
for the accuracy
of a diagnostic test. The AUC for the HM-Methyl-Light-Assay is: 0_844.
Figure 42 shows the Receiver Operating Characteristic curve (ROC curve) of the
SEQ
ID NO:35 -HM MethylLighta-Assay for Adenenomas according to EXAMPI.E
3(additional
sets of samples). The area under an ROC curve (AUC) is a raeasure for the
accuracy of a
diagnostic test. The AUC for the HM MethylLighi' "s-Assay is: 0.748.
Figura 43 shows the level of inethylation in different tumor and healthy
tissues
detertnined by a SEQ ID NO 35 -HeavyMet'hyllm MethyLightTM assay according to
example 4.
The Y-axis shows the degree of methylation within the region of the SEQ ID
NO:35 gene
investigated. Besides the colon cancer samples only one of the two breast
cancer tissues were
inethylated.
Figure 44 shows the level of inethyiation in different brcast cancer tissues
detiermined by
a SEQ ID NO:35 -HeavyMethyP MethyLightT" assay according to F.XAMPLE 4. Only
one
was methylated.
Figure 45 s hows the 1 evel o f inethylation i n s enim s amples d etennined
by a S EQ ID
NO:35 HeavyMethyP MethyLight " assay according to EXA1ViPLE 4. The Y-axis
shows the
degree of methylation within the region of the SEQ ID NO:35 gene investigated.
Fignre 46 shows the ROC curve of the SEQ ID NO:34 -MSP-Methyl-Light T""-Assay
according to EXAMPLE 9. The AUC is: 0.84.
Figure 47 shows the ROC curve of the SEQ ID NO:29 -MSP-Methyl-Light TM-Assay
CA 02528853 2005-12-23
aocording to EXAMPI.E 10. The AUC is: 0.80.
Figure 48 shows the regression plot of the percentage methylation within SEQ
ID NO:35
calculated in each sample using the MSP and HeavyMethyl T" variants of the
MethyLight T""
assay.
Figare 49 shows the ROC curve of the SEQ ID NO:29 -MSP-MethylLight TM-Assay
according to EXAMPLE 8 (first sample set). The AUC is: 0.93.
Figure 50 shows the ROC curve of the SEQ ID NO:29 -MSP-MethylLight Tm-Assay
according to BXAMPLE 8 (second sample set). The AUC is: 1.
Figure 51 shows the ROC curve of the SEQ ID NO:39 -MSP-MethylLight Tm-Assay
according to EXAMPLB 9. The AUC is: 0.94.
Figures 52 to 59 show the amplification of stool samples according to example
10. The
X-axis shows the cycle number of the polymerase chain reaction, the Y-axis
shows the delta Rn,
whieh is the fluorescence intensity over background, the horizontal line (A)
represents the
threshold level of the calibration curve (B), if the curve reaches above (A)
methylation has been
detected.
Figures 60 to 71 show the PCR amplification of samples according to example
11.Each
individual sample is annotated on the x-axis as 'n' denoting normal, or 't'
denoting tumour. A
bronchial tissue sample was accidentally analysed in some assays, these are
annotated with an
arrow, and are iYrelevant in the context of the present invention. The Y-axis
shows the level of
methylation ('methylation rate ') in the sample. The methylation rate
{methy2ated/(methylated
+ unmethylated) are shown for the fragments corresponding to the primer paus
in Table 4..
Figures 72 to 86 show the PCR amplification of samples according to assays I
to 15 of
example 14 respectively. The X-axis shows the cycle number of the polymerase
chain reaction,
the Y-axis shows the fluorescence intensity over background.
Figure 72 shows the amplification curve of assay 1.
Figure 73 shows the amplification curve of assay 2.
Figure 74 shows the amplification curve of assay 3.
Figure 75 shows the amplification curve of assay 4.
Figure 76 shows the amplification curve of assay 5.
Figure 77 shows the amplification curve of assay 6.
Figu.re 78 shows the amplification curve of assay 7.
16
CA 02528853 2005-12-23
Figare 79 shows the amplification cnrve of assay 8.
Figure 80 shows the amplification curve of assay 9.
Figure 81 shows the amplification curve of assay 10.
Figure 82 shows the amplification curve of assay 11.
Figure 83 shows the amplification curve of assay 12.
Figure 84 shows the amplification curve of assay 13.
Figure 85 shows the amplification curve of assay 14.
Figore 86 shows the amplification curve of assay 15.
Figures 87 to 95 show the class prediction performance of quadratic SVA+is
trained on the data set according to example 14. Circled points are the
support vectois defining
the borderline (white) between the lighter and darker shaded areas,
Figure 87 shows an SVM trained using Assays 12 and 9.
Figure 88 shows an SVM trained using Assays 12 and 11.
Fignre 89 shows an SVM traixud using Assays 12 and 15.
Figure 90 shows an SVM trained using Assays 7 and 6.
Figure 91 shows an SVM tsained using Assays 11 and 5.
Figure 92 shows an SVM trained using Assays 7 and 5.
Figure 93 shows an SVM trained using Assays 6 and 4.
Figure 94 shows an S'V"M trained using Assays 12 and 7.
Figure 95 shows an SVM trained using Assays 12 and 6.
Figure 96 shows a ranked matrices of data obtained according to Examples 1 and
2
according to CpG methylation differences between the two classes of tissues,
(aerodigestive
cancer on the left and healthy aerodigestive tissues on the right) using an
algorithim. The most
sigaificant CpG positions are at the bottom of the matrix with significance
decreasing towards
the top. Red indicates total methylation at a given CpG position, green
represents no
methylation at the particular position.. Each row represents one specific CpG
position within a
geae and esah column shows the methylation profile for the different CpGs for
one sample.
DETATLED DESCRIPTION OF THE;QWENTION
De ti Q-s:
The term "Observe3/Expected Ratio" ("O/E Ratio") refers to the frequency of
CpG
dinucleotides within a particular DNA sequeace, and corresponds to the
[niiinber of CpG sites 1
(number of C bases x number of G bases)] x band length for each fragment.
17
CA 02528853 2005-12-23
The term "CpG island" refers to a contigaous region of genonlic DNA that
satisfies the
rxiteria of (1) having a frequency of CpG dinucleotides corresponding to an
"Observed/Expected
Ratio" >0.6, and (2) having a "GC Content" >0.5. CpG islands are typically,
but not always,
between about 0.2 to about t kb, or to about 2kb in length.
The term "methylation state" or "methylation status" refers to the presence or
absence of
5-methylcytosine ("5~Cyt") at one or a plurality of CpG dinucleotides within a
DNA sequence.
Methylation states at one or more particular CpG methylation sites (each
having two CpG CpG
dinucleotide sequences) within a DNA sequence include "mmethylated," "fully-
methylated"'
and "hemi-methylated."
The term "hemi snethylation" or "hemimethylation" refers to the methylation
state of a
palindromic CpG methylation site, where only a single cytosine in one of the
two CpG
dinucleotide sequences of the palindromic CpG methylation site is methylated
(e.g., 5'-
CCMGG-3' (top strand): 3'-GGCC-5' (bottom strand)).
The term 'AUC' as used herein is an abbreviation for the area under a curve.
In
particular it refers to the area under a Receiver Operating Characteristic
(ROC) curve.'The ROC
curve is a plot of the true positive rate against the false positive rate for
the different possible
cutpoints of a diagnostic test. It shows the tradeoff between sensitivity and
specificity depending
on, the selected cutpoint (any increase in sensitivity will be accompanied by
a d'ecrease in
specificity). The area under an ROC curve (AUC) is a measure for the aeeuracy
of a diagnostic
test (the larger the area the better, optimum is 1, a random test would have a
ROC curve lying on
the diagonal with an area of 0.5; for reference: I.P. Egan. Signat Detection
Theory and ROC
Analysis, Academic Press, New York,1975).
The term "hypermethylation" refers to the average methylation state
corresponding to an
increased presence of 5-mCyt at one or a plurality of CpG dinucleotides within
a DNA sequence
of a test DNA sample, relative to the amount of 5-mCyt found at corresponding
CpG
dinucleotides within a nonnal control DNA sample.
The term "hypomethylation" refers to the average methylation state
corresponding to a
decreased presence of 5-mCyt at one or a plurality of CpG dinucleotides within
a DNA
sequence of a test DNA sample, relative to the amount of 5-mCyt found at
corresponding CpG
18
CA 02528853 2005-12-23
dinucleotides within a normal control DNA saniple.
The term "nicroarray" refers broadly to both "DNA microarrays," and 'DNA
chip(s),' as
recognized in the art, encompasses all art-recognized solid supports, and
encompasses all
methods for af.tixing nucleic acid molecules thereto or synthesis of nucleic
acids thereon.
"Genetic parameters" are mutations and polymorphisms of genes and sequences
fwrther
required for their regulafiion. To be designated as mutations are, in
particular, insertions,
deletions, point mutations, inveisions and polymoiphisms and, particularly
prefetxed, SNPs
(single nucleotide polymorphisms).
"Epigenetic parameters" are, in part.icular, eytosine methylations. Further
epigenetic
parameters include, for example, the acetylation of histones which, however,
cannot be directly
analyzed using the described method but which, in turn, correlate with the DNA
methylation.
The term "bisulfite reagent" refers to a reagent comprising bisulfite,
disulfite, hydrogen
sulfite or combinations thereof, usefnl as disclosed heroin to distinguish
between methylated and
unmethytated CpG dinucleotide sequences.
i5 The term '2Vlethylation assay" refers to any assay for determining the
methylation state
of one or more CpG dinucleotide sequences within a sequence of DNA.
The term "MS.AP-PCR" (Methylation-Sensitive .Arbitrarily-Primed Polylnerase
Chain
Reaction) refers to the art-recognized technology tbat allows for a global
scan of the genome
using CG-rich pruners to focus on the regions most likely to contain CpG
dinucleotides, and
described by Gonzalgo et al., Cancer Research 57:594599,1997.
The term "MethyLightTM" refers to the att-recognized IIuorescence-based real-
time PCR
technique described by Eads et al., Cancer Res. 59:2302-2306,1999.
The term "HeavyMethyTm" assay, in the embodiment thereof implemented herein,
refers
to an assay, wherein methylation specific bdocking probes (also referred to
herein as blockers)
covering CpG positions between, or covered by the amplification primers enable
methylation-
specific selective amplification of a nucleic acid sample.
The term "HeavyMethylTM MethyLight'' w' assay, in the embodiment thereof
implemented herein, refers to a HeavyMethylT"Y MethyLightTu assay, which is a
variation of the
MethyLightTM a ssay, wherein the MethyLightTu assay i s combined w ith m
ethylation specific
19
CA 02528853 2005-12-23
blocking probes covering CpG positions between the amplification primers.
The term "Ms-SNuPE" (Methylation-sensitive Single Nucleotide Primer Extension)
refers to the art-recogaixed assay described by Gonzalgo & Jones, Nucleic
Acids Res. 25:2529-
2531,1997.
The term "MSP" (Methylation-specific PCR) refers to the art-recognized
methylation
assay described by Herman et al. Proc. NatL Acad. Sci. USA 93:9821-9826, 1996,
and by US
Patent No. 5,786,146.
The term "COBRA ' (Combined Bisulfite Restriction Analysis) refers to the art-
recognized methylation assay described by Xiong & Laird, Nucleic Acids Res.
25:2532-2534,
1997.
The term "MCA" (Methylated CpG Island Amplification) refers to the methylation
assay
described by Toyota et al., Cancer Res. 59:2307-12, 1999, and in WO
00/26401A1.
The term "hybridizgtion" is to be understood as a bond of an oligonucleotide
to a
complementary sequence along the lines of the Watson-Crick base pairings in
the sample DNA,
forming a duplex strncture.
"Stringent hybridization conditions," as defined herein, involve hybridizing
at 68 C in .
5x SSC15x Denhardt's solution/1.0% SDS, and washing in 0.2x SSC/0.1% SDS at
room
temperature, or involve the art-recognized equivalent thereof (e.g.,
conditions in which a
hybridization is canied out at 60 C in 2.5 x SSC buffer, followed by several
washing steps at
37 C in a low buffer concentration, and reanains stable). Moderately stringent
conditions, as
defined herein, involve including washing in 3x SSC at 42 C, or the art-
recognized equivalent
thereof. The parameters of salt concentration and temperature can be varied to
achieve the
optimal level of identity between the probe and the target nucleic acid.
Guidance regard'zng such
conditions is available in the art, for example, by Sambrook et al., 1989,
Molecular Cloning, A
Laboratory Manual, Cold Spring Harbor Press, N.Y.; and Ausubel et al. (eds.),
1995, Current
Protocols in Molecular Biology, (John Wiley & Sons, N.Y.) at Unit 2.10.
The terms "array SEQ ID NO," "composite array SEQ ID NO," or "composite array
sequence" refer to a sequence, hypothetical or otherwise, consisting of a head-
to-tail (5' to 3')
linear composite of all individual contiguous sequences of a subject array
(e.g., a head-to-tail
CA 02528853 2005-12-23
composite of SEQ ID NOS:1-71, in that order).
The terms "array SEQ ID NO node," "composite array SEQ ID NO node," or
"composite array sequence node" refer to a junction between any two individual
contiguous
sequences of the "array SEQ ID NO," the "composite array SEQ ID NO," or the
"composite
array sequence."
In reference to composite array sequences, the phrase "contiguous nucleotides"
refers to
a contiguous sequence region of any individual contiguous sequence of the
composite array, but
does not include a region of the composite array sequence that includes a
"node," as defined
herein above.
v iew:
The present invention provides for molecular genetic markers that have novel
utility for
the analysis of methylation patterns associated with the development of
colorectal cell
proliferative disorders. Said markers may be used for detecting or
distinguishing between
colorectal cell proliferative disorders, thereby providing improved means for
the classification
and treatment of said disorders. In an alternative embodiment said molecular
genetic markers
may be used for the detection of aerodigestive cell proliferative disorders.
Bisulfite mod fication of DNA is an art-recognized tool used to assess CpG
methylation
status. 5-methylcytosine is the most frequent covalent base modification in
the DNA of
eukaryotic cells. It plays a role, for example, in the regulation of the
transcription, in genetic
imprinting, and in tumorigenesis. Therefore, the identification of 5-
methylcytosine as a
component of genetic information is of considerable interesi. However, 5-
methylcytosine
positions caamot be identified by sequencing, because 5-methylcytosine has the
same base
pairing behavior as cytosine. Moreover, the epigenetic information carried by
5-methylcytosine
is completely lost during, e.g., PCR amplification.
The most frequently used method for analyzing DNA for the presence of 5-
methylcytosine is based upon the specific reaction of bisulfite with cytosine
whereby, upon
subsequent alkaline hydrolysis, cytosine is converted to uracil which
corresponds to thymine in
its base pairing behavior. Significantly, however, 5-methylcytosine remains
unmodified under
these conditions. Consequently, the original DNA is converted in such a manner
that
methylcytosine, which originally could not be distinguished from cytosine by
its hybridization
behavior, can now be detected as the only remaining cytosine using standard,
art-recognized
molecular biological techniques, for example, by amplification and
hybridization, or by
21
CA 02528853 2005-12-23
sequencing. All of these techniques are based on differential base pairing
properties, which can
now be fully exploited.
The prior art, in terms of sensitivity, is defined by a method comprising
enclosing the
DNA to be analyzed in an agarose matrix, thereby preventing the diffusion and
renaturation of
the DNA (bisulfite only reacts with single-stranded DNA), and replacing all
precipitation and
purification steps with fast dialysis (Olek A, et al., A modified and improved
method for
bisulfite based cytosine methylation analysis, Nucleic Acids Res. 24:5064-6,
1996). It is thus
possble to analyze individual cells for methylation status, illushating the
utility and sensitivity
of the naethod. An ovelview of art-recognized methods for detecting 5-
methylcytosine is
provided by Rein, T., et al., Nucleic Acids Res., 26:2255, 1998.
The bisulfite technique, barring few exceptions (e.g., Zeschnigk M, et a l.,
Eur J Hum
Gerud. 5:94-98, 1997), is cumently only used in research. Iu all instances,
short, specific
fragments of a known gene are amplified subsequent to a bisulfite treatment,
and either
completely sequenced (Olek & Walter, Nat Genet. 1997 17:275-6, 1997),
subjected to one or
more primer extension reactions (Gonzalgo & Jones, Nucleic Acids Res., 25:2529-
31, 1997; WO
95/00669; U.S. Patent No. 6,251,594) to analyze individual cytosine positions,
or treated by
enzymatic digestion (Xiong & Laird, Nucleic Acids Res., 25:2532-4, 1997).
Detection by
hybridization has also been described in the art (Olek et al., WO 99/28498).
AdditionaIIy, use of
the bisulfite technique for methylation detection with respect to individual
genes has been
described (Grigg & Clark, Bioessays, 16:431-6, 1994; Zeschnigk M, et al., Hum
Mol Genet.,.
6:387-95, 1997; Feil R, et al., Nucleic Acids Res., 22:695-, 1994; Martin V,
et al., Gene,
157:261-4, 1995; WO 9746705 and WO 9515373).
The p resent i nvention p rovides for the use o f t he bisulfite t echnique ,
in c ombination
with one or more methylation assays, for determination of the methylation
status of CpG
dinuclotide sequences within sequences from the group consisting of SEQ ID
NOS:1 to SEQ ID
NO:64. According to the present invention, detennination of the methylation
status of CpG
dinuclotide sequences within sequences from the group consisting of SEQ ID
NOS:1 to SEQ ID
NO:64 has diagnostic and progaostic utility.
Methylation Assay Procedures. Various methylation assay procedures are known
in the
art, and can be used in conjunction with the present inveation. These assays
allow for
detertnination of the methylation state of one or a plurality of CpG
dinucleotides (e.g., CpG
islands) within a DNA sequence. Such assays involve, among other techniques,
DNA
sequencing of bisulfita-treated DNA, PCR (for sequence-specific
amplification), Southern blot
analysis, and use of inethylation-sensitive restriction enzymes.
22
CA 02528853 2005-12-23
For example, genomic sequencing has been simplified for analysis of DNA
methylation
pattecns and 5-methylcytosine distribution by using bisulfite treatment
(Frommer et al., Proc.
Natl. Acad. Sci. USA 89:1827-1831, 1992). Additionally, restriction enzyme
digestion of PCR
products amplified from bisulfite-converted DNA is used, e.g., the method
described by Sadri &
Hornsby(Nucl. Acids Res. 24:5058-5059, 1996), or COBRA (Combined Bisulfite
Restriction
Analysis) (Xiong & Laird, Nucleic Acids Res. 25:2532-2534,1997).
COBRA. COBRAm analysis is a quantitative methylation assay useful for
detennining
DNA methylation levels at specific gene loci in small amounts of genomie DNA
(Xiong &
Laird, Nucleic Acids Re<r. 25:2532-2534, 1997). Briefly, restriction enzyme
digestion is used to
reveal methylation-dependent sequence differences in PCR products of sodium
bisulfite-treated
DNA. Methylation-dependent sequence differences are first introduaed into the
genomic DNA
by standard bisulfite treatmerrt aeeording to the procedure described by
Frommer et al. (Proc.
Natl. Acad Sci. USA 89:1827-1831, 1992). PCR amplification of the bisulfite
converted DNA
is then performed using primers specific for the CpG islands of interest,
followed by restriction
endonuclease digestion, gel electrophoresis, and detection using specific,
labeled hybridization
probes. Methylation levels in the original DNA sample are represented by the
relative amounts
of digested and undigested PCR product in a linearly quantitative fashion
across a wide
spectrum of DNA methylation levels. In addition, this technique can be
reliably applied to DNA
obtained from microdissected paraffin-embedded tissue samples.
Typical reagents (e.g., as might be found in a typical COBRAm-based kit) for
COBRAm analysis may include, but are not limited to: PCR primers for specific
gene (or
bisulfite treated DNA sequence or CpG island); restriction enzyme and
appropriate buffer; gene-
hybridization oligo; control hybridization oligo; kinase labeling Idt for
oligo probe; and labeled
nucleotides. Additionally, bisulfite conversion reagents may iuclude: DNA
denaturation buffer;
sulfonation buffer. DNA recovery reagents or kits (e.g., precipitation,
ultrafiltration, affmity
column); desulfonation buffer; and DNA recovery components.
Preferably, assays such as "MethyLight'w' (a fluorescence-based real-time PCR
technique) (Eads et al., Cancer Res. 59:2302-2306, 1999), Ms-SNnPE7m
(Methylation-sensitive
Single Nucleotide Primer Extension) reactions (Gonzalgo & Jones, Nucleic Acids
Res. 25:2529-
2531, 1997), methyla6on-speci5c PCR ("MSP '; Herman et al., Proc. Natt. Acad.
Sci. USA
93:9821-9826, 1996; US Patent No. 5,786,146), and methylated CpG island
amplification
{"MCA"; Toyota et al., Cancer Res. 59:2307-12, 1999) are used alone or in
combination with
other of these methods.
MethyLight TM. The MethyLight "" assay is a high-throughput quantitative
methylation
23
CA 02528853 2005-12-23
assay that utilizes fluorescence-based real-time PCR (Ta4MMap TM) technology
that requirea no
fiuther manipulations after the PCR step (Eads et al., Cancer Res. 59:23022-
2306, 1999).
Briefly, the MethyLight''"' process begins with a mixed sample of genomic DNA
that is
converted, in a sodium bisulfite reaction, to a mixed pool of methylation-
dependent sequence
differences according to standard procedures (the bisulfite process converts
unmethylated
cytosine residues to uracil). Fluorescence-based PCR is then performed either
in an "anbiased"
(with primers that do not overlap known CpG methylation sites) PCR reaction,
or in a"biased"
(with PCR primers that overlap known CpG dinucleotides) reaction. Sequence
discrimination
can occur either at the level of the amplification process or at the level of
the fluorescence
detection process, or both.
The ArlethyLight'"'' assay may be used as a quantitative test for methylation
patterns in
the genomic DNA sample, wherein sequence discrimination occurs at the level of
probe
hybridization. In this quantitative version, the PCR reaction provides for
unbiased amplification
in the presence of a fluorescent probe that overlaps a particular putative
methylation site. An
unbiased control for the amount of input DNA is provided by a reaction in
which neither the
primers, nor the probe overlie any CpG dinucleotides. Alternatively, a
qualitative test for
genomic methylation is achieved by probing of the biased PCR pool with either
control
oligonucleotides ttiat do not "cover" known methylation sites (a
fluorescencabased version of
the '1VbSP" technique), or with oligonucleotides covering potential
methylation sites.
The MathyLight TM process can by used with a"TaqMan " probe in the
amptification
process. For example, double-sttanded genomic DNA is treated with sodium
bisulfite and
subjected to one of two sets of PCR reactions using TaqMan probes; e.g., with
either biased
primers and TaqMan probe, or unbiased primers and TaqMan probe. The TaqMan*
probe
is dual-labeled with fluorescent 'reporter" and "quenchee' molecules, and is
designed to be
Z5 specific for a relatively high GC content region so that it melts out at
about 10 C higher
temperature in the PCR cycle than the forward or reverse primers. This allows
the TaqMan
probe to remain fully hybridized during the PCR annealinglextension step. As
the Taq
polymerase enzymatically synthesizes a new strand during PCR, it will
eventuaUy reach the
annealed TaqMan probe. The Taq polymerase 5' to 3' endonuclease activity will
then
10 displace the TaqMan probe by digesting it to release the fluorescent
reporter molecule for
quantitative detection of its now unquenched signal using a real-time
fluorescent detection
system..
Typical reagents (e.g., as might be found in a typical MethyLight Ta-based
kit) for
MethyLight;"' analysis may include, but are not limited to: PCR primers for
specific gene (or
24
CA 02528853 2005-12-23
bisulfite treated DNA sequence or CpG island); TaqMan probes; optimized PCR
buffers and
deoxynucleotides; and Taq polymerase.
Ms-SNuPE. The Ms-SNuPETm technique is a quantitative method for assessing
methylation differences at specific CpG sites based on bisulfite treatment of
DNA, followed by
single-nucleotide primer extension (Gonzalgo & Jones, Nucleic Acids Res.
25:2529-2531, 1997).
Briefly, genoniic DNA is reacted with sodium bisulfite to convert unmethylated
cytosine to
uracil while leaving 5-methylcytosine unchanged. Amplification of the desired
target sequence
is then performed using PCR primers specific for bisulfite-converted DNA, and
the resulting
product is isolated and used as a template for methylation analysis at the CpG
site(s) of interesk
Small amounts of DNA can be analyzed (e.g., microdissected pathology
sections), and it avoids
utilization of restriction enzymes for determining the methylation status at
CpG sites.
Typical reagents (e.g., as might be found in a typical Ms-SNuPEm-based kit)
for Ms-
SNuPEm analysis may include, but are not limited to: PCR primers for specific
gene (or
bisulfite treated DNA sequence or CpG island); optimized PCR buffers and
deoxynucleotides;
gel extraction Iat; positive control primers; Ms-SNuPEm primers for specific
gene; reaction
buffer (for the Ms-SNuPE reaction); and labelled nucleotides. Additionalty,
bisuifite conversion
reagents may include: DNA denaturation buffer; sulfonation buffer; DNA
recovery regents or
Idt (e.g., precipitation, ultrafiltration, affinity column); desulfonation
buffer; and DNA recovery
components.
MSP. MSP (methylation specific PCR) allows for assessing the methylation
status of
virtually any group of CpG sites within a CpG island, independent of the use
of methylation-
sensitive restriction enzymes (Herman et al. Proc. Natl. Acad. Sci. USA
93:9821-9826,1996; US
Patent No. 5,786,146). Briefly, DNA is modified by sodium bisulfite converting
all
unmethylated, but not methylated cytosines to uracil, and subsequently
amplified with primers
specific for methylated versus umnethylated DNA. MSP requires only small
quantities of DNA,
is sensitive to 0.1% metlhylated alleles of a given CpG island locus, and can
be performed on
DNA extracted from paraffin-embedded samples. Typical reagents (e.g., as might
be found in a
typical MSP-based Icit) for MSP analysis may include, but are not limited to:
methylated and
unnaethylated PCR primers for specific gene (or bisulfite treated DNA sequence
or CpG island),
optimized PCR buffers and deoxynucleotides, and specific probes.
MCA. The MCA technique is a method that can be used to screen for altered
CA 02528853 2005-12-23
methylatioai patteans in genomic DNA, and to isolate specific sequences
associated with these
changes (Toyota et al., Cancer Res. 59:2307-12, 1999). Briefly, restriction
enzymes with
different sensitivities to cytos;ne methylation in their recognition sites are
used to digest
genomic DNAs from primary tumors, cell lines, and normal tissues prior to
arbitrarily primed
PCR amplification. Fragments that show differential methylation are cloned and
sequenced
after resolving the PCR products on lugh-resolution polyacrylamide gels. The
cloned fragments
are then used as probes for Southem analysis to confirm differential
methylation of these
regions. Typical reagents (e.g., as might be found in a typical MCA-based kit)
for MCA
analysis may include, but are not limited to: PCR primers for arbitrary
priming Genomic DNA;
PCR b uffers and nucleotides, restriction enzymes and appropriate b uffers; g
ene hybridization
oligos or probes; control hybridization oligos or probes.
Genpmic Seau(mce$ Acg:ording to SEO ID NO:1 to SEO ID NO:64. Md Non-naturallv
Occuring
Treated Variants Thereof Accordins t o S EO ID NOS:30m to $ EO ID NQS:535 and
S EO ID
NM:65 TO SEO ID NO:88. were Determined to have NoveLUtilitv fo; the Detection.
Classification and/or Treatment of Colorec~ Cell Proliferative Disorders
In one embodiment the invention provides a method for detecting and/or for
detecting
and distingtushing between or among colon cdl proliferative disorders in a
subject. Said method
comprises the following steps: i) contacting genomic DNA isolated from bodily
fluids obtained
from the subject with at least one reagent, or seaies of reagents that
distingaishes between
methylated and non-methylated CpG dinucleotides within at least one targa
region of the
genomic DNA, wherein said contiguous nucleotides comprise at least one CpG
d'mucleotide
sequence; and ii) detecting, or detecting and distinguishing between or among
colon cell
proliferative disorders afforded with a sensitivity of greater than or equal
to 80% and a
specificity of greater than or equal to 80'/o.
Preferably, the sensitivity is from about 75% to about 96%, or from about 80%
to about
90%, or from about 80% to about 85%. Perferably, the specificity is is from
about 75% to about
96o/Q, or fiom about 80% to about 90%, or from about 80% to about 85%.
Genomic DNA may be isolated by any means standard in the art, including the
use of
cammercially available kits. Briefly, wherein the DNA of interest is
encapsulated in by a
cellular membrane the biological sample must be disrupted and lysed by
enzymatic, chelnical or
mechanical means. The DNA solution may then be cleared ofproteins and other
contaminants,
e.g., by digestion with proteinase K. The genomic DNA is then recovered from
the solution.
26
CA 02528853 2005-12-23
T"his may be carried out by means of a variety of methods including salting
out, organic
extraction or binding of the DNA to a solid phase support. The choice of
method will be
affected by several factors including time, expense and required quantity of
DNA. Body Quids
are the preferred source of the DNA; particularly preferred are blood plasma,
blood serum,
whole blood, isolated blood cells and cells isolated from the blood.
The genomic DNA sample is then treated in such a mamier that cytosine bases
which are
unmethylated at the 5'-position are converted to uracil, thymine, or another
base which is
dissimilar to cytosine in terms of hybridization behavior. This will be
understood as 'treatment'
herein.
The above desaribed treatment of genomic DNA is preferably carried out with
bisulfite
(hydrogen sulfite, disulfite) and subsequent alkaline hydrolysis which results
in a conversion of
non-methylated cytosine nucleobases to uraciI or to another base which is
dissimilar to cytosine
in terms of base pairing behavior.
The treated DNA is then analysed in order to detenmine the methylation state
of one or
more target gene sequences (prior to the treatment) associated with the
development of
colorectal carcinoma. It is particularly preferred that the target region
comprises, or hybridizes
under stringent conditions to at least 16 contiguous nucleotides of at least
one gene or sequence
selected from the group consisting the genes and sequences as listed in Table
1. It is fmther
preferred that the sequences of said genes in Table 3 as described in the
accompanying sequence
listing are analysed. The method of analysis may be selected from those known
in the art,.
including those listed herein. Particularly preferred are Methy.Light, MSP and
the use of
blocking oligonucleotides as will be descn'bed herein. It is further preferred
that any
oligonucleotides used in such analysis (including primers, blocking
oligonucleotides and
detection probes) should be reverse complementary, identical, or hybridize
under stringent or
higlily stringent conditions to an at least 16-base-pair long segment of the
base sequences of one
or more of SEQ ID NO: (IDBISEQFIRST) to SEQ ID NO: and (IDBISEQI.AST) and
sequences
complementary thereto.
Aberrant methylation, more preferably hypermethylation of one or more genes or
genomic sequences taken from the group consisting of: SEQ ID NOS:1-3, 14, 16,
21, 22, 23, 44-
46, 48, 50, 59-64, COX7B, CSTS, EYA3, FTH1, RNF4, SOX 21, RNF4, ALX 4, ONC2,
NGFR, DLX-5, HVIAAT, BTF3 Homolog 1, ADCY9, BCL-5, BCOR, TAFII28, HOMEOBOX
PROTEIN SIX6, GSK-3 BETA, ELK-L, HLA-F, TGFBR2, AR, RNF30 and DUX4 are
associated with the presence of colorectal carcinoma.
Analysis of one or a plurality of the sequences enables for the first time
detecting, or
27
CA 02528853 2005-12-23
detecting and distinguishing between or among colon cell proliferative
disorders afforded with a
sensitivity of greater than or equal to 80% and a specificity of greater than
or equal to 80%.
Sensitivity is calculated as: {detected colon neoplasia/all colon neoplasia);
and specificity is
calculated as (non-detected negatives/total negatives).
S Preferably, the sensitivity is from about 75% to about 96%, or from about
80% to about
90%, or from about 80% to about 85%. Perferably, the specificity is is from
about 75% to about
96%, or from about 80% to about 90%, or from about 80% to about 85%.
Colon neoplasia can be defined as aIl colon malignancies or colon malignancies
and
large (> 1 cm adenomas), or subsets thereo~ Negatives can be defined as any
disease other than
colon malignancy and adenomas, or healthy individuals.
In one embodiment the method discloses the use of one or more genes or genomic
sequences selected from the group consisting SEQ ID NOS:1-3, 14, 16, 21, 22,
23, 44-46, 48,
50, 59-64, COX7B, CST5, EYA3, FTHI, RNF4, SOX 21, RNF4, AIX 4, ONC2, NGFR, DLX-
5, HVIAAT, BTF3 Homolog 1, ADCY9, BCL-5, BCOR, TAFII28, HOMEOBOX PROTEIN
SIX6, GSK-3 BETA, ELK L, HLA F, TGFBR2, AR, RNF30 and DUX4 as markers for the
differentiation, detection and distinguishing of colon cell proliferative
disorders.
Said use of the gene may be enabled by means of any analysis of the expression
of the
gene, by means of mRNA expression analysis or protein expression analysis.
However, in the
most preferred embodiment of the imrention, the detection, differentiation and
distinguishing of
colon cell proliferative disorders is enabled by means of analysis of the
methylation status of one
or more genes or genomic sesluences selected from the group consisting SEQ IL)
NOS:1-3, 14,
16, 21, 2 2,2 3, 4 4-46, 4 8, 5 0, 5 9-64, C OX7B, C ST5, EYA3, F TH1, RNF4, S
OX 21, RNF4,
ALX 4, ONC2, NGFR; DLX-5, HVIAAT, BTF3 Homolog 1, ADCY9, BCL-5, BCOR,
TAFII28, HOMBOBOX PROTEIN SIX6, GSK-3 BETA, ELK-L, HLA-F, TGFBR2, AR,
RNF30 and DUX4 and their promoter or regulatory elements. Methods for the
methylation
analysis of genes are descn'bed herein,
Abezrant levels of mRNA expression of the genes, genoniic sequences or genes
regulated
by genomic sequences according to SEQ ID NOS: 1-3, 14, 16, 21, 22, 23, 44-46,
48, 50, 59-64,
COX7B, CSTS, EYA3, FTHI, RNF4, SOX 21, RNF4, ALX 4, ONC2, NGFR, DLX-5,
HVIAAT, BTF3 Homolog 1, ADCY9, BCL-5, BCOR, TAFII28, HOMEOBOX PROTEIN
SIX6, GSK-3 BETA, ELK L, HLA-F, TGFBR2, AR, RNF30 and DUX4 accozrling to Table
3
are associated with colorectal carcinoma.
Accordingly, increased or decreased levels of expression of said genes or
sequences are
associable with the development of colorectal carcinoma and other colorectal
ceIl proliferative
28
CA 02528853 2005-12-23
disorders.
To detect the presence of mRNA encoding a gene or genomic sequence in a
detection
system for colon cancer, a sample is obtained from a patient. The sample can
be a tissue biopsy
sample or a sample of blood, plasma, sernm or the like. The sample may be
treated to extract
the nucleic acids contained therein. The resulting nucleic acid from the
sample is subjected to
gel electrophoresis or other separation techniques. Detection involves
contacting the nucleic
acids and in particular the mRNA of the sample with a DNA sequence serving as
a probe to
form hybrid duplexes. The stringency of hybridisation is detennined by a
number of factors
during hybridisation and during the washing procedure, including temperature,
ionic strength,
length of time and concentration of formamide. These factors are outlined in,
for example,
Sambrook e t a 1. ( Molecular C loning: A Laboratory M anual, 2 d ed., 1989).
D etection o f t he
resulting duplex is usually accomplished by the use of labelled probes.
Alternatively, the probe
may be unlabeled, but may be detectable by specific binding with a ligand
which is labelled,
either directly or indirectly. Suitable labels and methods for labelling
probes and ligands are
known in the art, and include, for example, radioactive labels which may b e
incorporated by
known methods (e.g., nick translation or kinasing),biotin, fluorescent groups,
chemiluminescent
groups (e.g., dioxetanes, particulariy triggered dioxetanes), enzymes,
antibodies, and the like.
In o rder t o i ncrease the sensitivity o f t he d etection i n a sample o f m
RNA t ranscribed
from the gene or genomic sequence, the technique of reverse
tr=scription/polymerisation chain
reaction can be used to a mplify c DNA transcribed from the mRNA. The method
of reverse
t=anscription /PCR is well known in the art (for example, see Watson and
Fleming, supra).
The reverse transcription /PCR method can be performed as follows. Total
cellular RNA
is isolated by, for example, the standard guanidium isothiocyanate method and
the total RNA is
reverse transcribed. The reverse transcription method involves synthesis of
DNA on a template
of. RNA using a reverse transcriptase enzyme and a 3' end primer. Typically,
the primer
contains an oligo(dT) sequence. The cDNA thus produced is then amplified using
the PCR
method and EYA4 specific primers. (Belyavsky et al, Nucl Acid Res 17:2919-
2932, 1989; Krug
and Berger, Methods in Enzymology, Academic Press,N.Y., Vol.152, pp. 316-325,
1987 which
are incorporated by reference).
The present invention may also be described in certain embodiments as a kit
for use in
detecting a colon cancer disease state through testing of a biological sample.
A representative
lait may comprise one or more nucleic acid segments that selectively hybridise
to the mRNA and
a container for each of the one or more nucleic acid segments. In certain
embodiments the
nucleic acid segments may be combined in a single tube. In further
embodiments, the nucleic
29
CA 02528853 2005-12-23
acid segments may also include a pair of primers for amplifying the target
mRNA. Such Idts
may also include any buffers, solutions, solvents, enzymes, nucleotides, or
other components for
hybridisation, amplification or detection reactions. Preferred Isit components
include reagents
for reverse transcription-PCR, in situ hybridisation, Northera analysis and/or
RPA
The present invention finther provides for methods to detect the preseace of
the
polypeptide encoded by said genes or gene sequences in a sample obtained from
a patient.
Aberrant levels of polypeptide expression of the polypeptides encoded by the
genes,
genomic sequences or genes regulated by genomic sequences according to SEQ ID
NOS:1-3,
14,16, 21, 22, 23, 44-46, 48, 50, 59-64, COX7B, CSTS, EYA3, FTH1, RNF4, SOX
21, RNF4,
AL.X 4, ONC2, NGFR, DLX 5, HVIAAT, BTF3 Homolog 1, ADCY9, BCL-5, BCOR,
TAFII28, HOMEOBOX PROTEIN SIX6, GSK-3 BETA, ELK L, HLA-F, TGFBR2, AR,
RNF30 and DUX4 according to Table 3 are associated with colorectal carcinoma.
Accordingly over or under expression of said polypeptides are associable with
the
development of colorectal carcinoma and other colorectal cell proliferative
disorders.
Any method known in the art for detecting proteins can be used. Such methods
include,
but are not limited to immunodiB'usion, immunoelectrophoresss, immunochanical
methods,
binder-ligand assays, immunohistochemical techniques, agglutination and
complement assays..
(e.g., see B asic and Clinical I mmunology, Sites and Terr, e ds., Appleton &
L ange,. Norwalk,
Conn. pp 217-262, 1991 whieh is incorporated by reference). Preferred are
binder-ligand
immunoassay methods including reacting antibodies with an epitope or epitopes
and.
competitively displaaing a labelled protein or derivative thereof.
Certain embodiments of the present invention comprise the use of antibodies
specific to
the polypeptide encoded by the genes or genomic sequences of the group
consisting of SEQ lD
NOS:1-3, 14, 16, 21, 22, 23, 44-46, 48, 50, 59-64, COX7B, CST5, EYA3, FTH1,
RNF4, SOX
21, RNF4, ALX 4, ONC2, NGFR, DLX-5, HVIAAT, BTF3 Homolog 1, ADCY9, BCL-5,
BCOR, T.AFII28, HOMEOBOX PROTEIN SIX6, GSK-3 BETA, ELIC L, HT.A F, TGFBR2,
AR,12NF30 and DUX4.
Such antibodies may be useful for diagnostic and prognostic applications in
detecting the
disease state, by comparing a patient's levels of colon disease marker
expression to expression
of the same markers in normal individuals. In certain embodiments production
of monoclonal or
polyclonal antibodies can be induced by the use of the coded polypeptide as
antigene. Such
amibodies may in tum be used to detect expressed protsins as markers for human
disease states.
The levels of such proteins present in the peripheral blood or tissue sample
of a patient may be
quantifxed by conventional methods. Antibody-protein binding may be detected
and quantified
CA 02528853 2005-12-23
by a variety of means known in the art, such as labelling with fluorescent or
radioactive ligands.
The invention fiuther comprises ldts for performing the above-mentioned
procedures, wherein
such kits contain antibodies specific for the investigated polypeptides.
Numerous competitive and non-competitive protein binding immunoassays are well
known in the art. Antibodies employed in such assays may be unlabeled, for
example as used in
agglutination tests, or labelled for use a wide variety of assay methods.
Labels that can be used
include radionuclides, enzymes, fluorescers, chemiluminescers, enzyme
substrates or co-factors,
enzyme intnbitors, particles, dyes and the like for use in radioimmunoassay
(RIA), enzyme
immunoassays, e.g., enzyme-linked immunosorbent assay (ELISA), fluorescent
immunoassays
and the like. Polyclonal or monoclonal antibodies or epitopes thereof can be
made for use in
immunoassays by any of a number of methods known in the art.
One approach for preparing antibodies to a protein is the selection and
preparation of an
amino acid sequence of all or part of the protein, chemically synthesising the
sequence and
injecting it into an appropriate animal, usually a rabbit or a mouse (Milstein
and Kohler Nature
256:495-497,1975; Gulfre and Milstein, Methods in Enzymology: Immunochemical
Techniques
73:1-46, Langone and Banatis eds., Academic Press, 1981 which are incorporated
by reference).
Methods for preparation of the polypeptides or epitopes thereof include, but
are not limited to
chemical synthesis, recombinant DNA techniques or isolation from biological
samples.
In addition to the embodiments above wherein the methylation analysis of genes
or
genomic sequences selected from the group SEQ ID NOS:1-3, 14, 16, 21, 22, 23,
44-46, 48, 50,
59-64, COX7B, CST5, EYA3, FTH1, RNF4, SOX 21, RNF4, ALX 4, ONC2, NGFR, DLX-5,
HVIAAT, BTF3 Homolog 1,ADCY9, BCL-5, BCOR, TAF'IT28, HOMEOBOX PROTEIN
SIX6, GSK-3 BETA, ELK-L, HLA-F, TGFBR2, Alt, RNF30 and DUX4 according to Table
3
are analysed, the invention presents two further panels of genes with novel
utility for the
detection of colorectal cancer.
In a first further embodiment the present invention is based upon the analysis
of
methylation levels within one or more genes or genomic sequences taken from
the group
consisting of SEQ ID NOS:1-3, 14, 16, 21, 22,23, 44-46, 48, 50, 59-64, COX7B,
CST5, EYA3,
FTH1, RNF4, SOX 21, RNF4, ALX 4, ONC2, NGFR, DLX-5, HVIA.AT, BTF3 Homolog 1,
ADCY9, BCL-5, BCOR,TAFII28, HOMEOBOX PROTEIN SIX6, GSK-3 BETA, ELK-L,
HLA-F, TGFBR2, AR, RNF30, DUX4, SMAD7, GTMBP, EYA4 and TCF1-ALPõlIA
according to Table 3 and/or their regulatory sequences.
It is further preferred that the sequences of said genes or genomic sequences
are as
according to Table 3.
31
CA 02528853 2005-12-23
In a fiuther embodiment the present invention is based upon the analysis of
inethylation
levels within two or more genes or genomic sequences taken from the group
consisting all genes
and genomic sequences according to Table 3 andlor their regulatory sequences.
It is further
preferred that the sequences of said genes or genomic sequences are as
according to SEQ ID
NOS:1 to SEQ ID NO:64.
Particular embodiments of the present invention provide a novel application of
the
analysis of inethylation levels and/or pattems within said sequences that
enables a precise
detection, characterisation and/or treatment of colorectal cell proliferative
disorders. Early
detection of colorectal cell proliferative disorders is directly linked with
disease prognosis, and
the disclosed method thereby enables the physician and patient to make better
and more
informed treatment decisions. The sequences of the group consisting SEQ ID
NOS: 20, 41, 35,
3, 39, 33 & 27 have fiuther utility for the detection of arod.igestive cell
proliferative disorders.
FURTHER IMPROVE~VIENTS
The present invention provides novel uses for genomic sequences selected from
the
group consisting of SEQ ID NOS:1 to SEQ ID NO:64. Additional embodiments
provide
modified variants of SEQ ID NOS:1 to SEQ ID NO:64, as well as oligonucleotides
and/or PNA-
oligomers for analysis of cytosine methylation pattems within SEQ ID NOS:1 to
SEQ ID
NO:64.
An objective of the invention comprises analysis of the methylation state of
one or more-
CpG dinucleotides within at least one of the genomic sequences selected from
the group
consisting of SEQ ID NOS:1 to SEQ ID NO: 64 and sequences complementary
thereto.
The disclosed invention provides treated nucleic acids, derived from genomic
SEQ ID
NOS:I to SEQ ID NO:64, wherein the treatment is suitable to convert at least
one unmethylated
cytosine base of the genomic DNA sequence to uracil or another base that is
detectably
dissimilar to cytosine in terms of hybridization. The genomic sequences in
question may
comprise one, or more, consecutive or random methylated CpG positions. Said
treaiment
preferably comprises use of a reagent selected from the group consisting of
bisulfite, hydrogen
sulfite, disulfite, and combinations thereof. In a preferred embodiment of the
invention, the
objective comprises analysis of a non-naturally o ccurring modified nucleic
acid comprising a
sequence of at least 16 contiguous nucleotide bases in length of a sequence
selected from the
32
CA 02528853 2005-12-23
group consisting of SEQ ID NOS:304 to SEQ ID NO:535 and SEQ ID NOS:65 to SEQ
ID
NO:88, wherein said sequence comprises at least one CpG, TpA or CpA
dinucleotide and
sequences oomplementary thereto. The sequences of SEQ ID NOS:304 to SEQ ID
NO:535 and
SEQ II) NOS:65 to SEQ ID NO:88 provide non-naturally occurring modified
versions of the
nucleic acid according to SEQ ID NOS:1 to SEQ ID NO:64, wherein the
modification of each
genomic sequence results in the synthesis of a nucleic acid having a sequence
that is unique and
distinct from said genomic sequence as fbllows. For each sense strand genomic
DNA, e.g., SEQ
ID NO:1, four converted versions are disclosed. A first version wherein "C" -
+"T," but "CpG"
remains "CpG" (i.e., corresponds to case where, for the genomic sequence, all
"C" residues of
CpG dirnicleotide sequences are methylated and are thus not converted); a
second version
discloses the complement of the disclosed genomic DNA sequence (i.e. antisense
strand),
wherein "C" but 'CpG" remains "CpG ' (i.e., coiresponds to case where, for
all "C"
residues of CpG dinucleotide sequences are methylated and are thus not
converted). The
'upmethylated' oonverbed sequences of SEQ ID NOS:1 to SEQ ID NO:64 correspond
to SEQ ID,
NOS:65 to 76 and SEQ ]D NOS:304 to SEQ ID NO:419. A third chemically converted
version
of each genomic sequences is provided, wherein for all"C" residues, including
those =
of "CpG" dinucleotide sequences (i.e., corresponds to case where, for the
genornic sequences,
all "C" residues of CpG dinucleotide sequences are unmethylated); a final
chemically converted
version of each sequence, discloses the complement of the disclosed genomic
DNA sequence
(i.e. antisense strand), wherein "C" -+"T" for all "C" residues, including
those of "CpG"
dinucleotide sequences (i.e., corresponds to case where, for the complement
(antisense strand)
of each genomic sequence, all "C" residues of CpG dinucleotide sequences are
umneihylated).
Tbe 'dowinnethylated' converted sequences of SEQ ID NOS:1 to SEQ ID NO:64
correspond to
SEQ ID NOS: 77 to 88 and SEQ ID NOS:420 to SEQ ID NO:535.
Signifieantly, heretofore, the nucleic acid sequences and molecules according
SEQ ID
NOS:1-7, 9-11, 14-23, 27, 31, 32, 40-42, 44-46, 48, 50-55 and 59-64 were not
implicated in or
connected with the detection, classification or treatment of colorectal cell
proliferative disorders
or aerodigestive ce11 proliferative disorders..
In an alternative preferred embodiment, such analysis comprises the use of an
33
CA 02528853 2005-12-23
oligonucleotide or oligomer for detecting the cytosine methylation state
within genomic or
treated (chemically m odified) D NA, according t o S EQ ID NOS:1 t o S EQ ID N
0:53 S. S aid
oligonucleotide or oligomer comprising a nucleic acid sequence having a length
of at least nine
(9) nucleotides which hybridizes, under moderately stringent or stringent
conditions (as defined
herein above), to a treated nucleic acid sequence according to SEQ ID NOS: 65
to SEQ ID NO:
88 and SEQ ID NOS:304 to SEQ ID NO:535 and/or sequences complementazy thereto,
or to a
genomic sequence according to SEQ ID NOS:1 to SEQ ID N0:64 andlor sequences
complementary thereto.
Thus, the present invention includes nucleic acid molecules (e.g.,
oligonucleotides and
peptide nucleic aeid (PNA) molecules (PNA-oligomers)) that hybridize under
moderately
stringent and/or stringent hybridization conditions to all or a portion of the
sequences SEQ ID
NOS:1 to SEQ ID NO:535, or to the complements thereof. The hybridizing portion
of the
hybridizing nucleic acids is typically at least 9, 15, 20, 25, 30 or 35
nucleotides in length.
However, longer molecules have inventive utility, and are thus within the
scope of the present
invention.
Preferably, the hybridizing portion of the inventive hybridizing nucleic acids
is at least
95%, o r a t least 98%, or 100% i dentical t o the sequence, o r t o a p
ortion t hereof o f S EQ ID
NOS:1 to SEQ ID NO:535, or to the complements thereof.
Hybridizing nucleic acids of the type descnbed herein can be used, for e
xample, as a
primer (e.g., a PCR primer), or a diagnostic and/or prognostic probe or
primer. Preferably,
hybridization of the oligonucleotide probe to a nucleic acid sample is
performed under stringent
conditions and the probe is 100% identical to the target sequence. Nucleic
acid duplex or hybrid
stability is expressed as the melting temperature or Tm, which is the
temperature at which a
probe dissociates from a target DNA. This melting temperature is used to
define the required
stringency conditions.
For target sequences that are related and substantially identical to the
con:esponding
sequence of SEQ ID NOS:1 to SEQ ID NO:64 (such as allelic variants and SNPs),
rather than
identical, it is useful to first establish the lowest temperature at which
only homologous
hybridization occurs with a particular concentration of salt (e.g., SSC or
SSPE). Then, assuming
34
CA 02528853 2005-12-23
that 1% mismatching results in a 1 C decrease in the Tm, the temperature of
the final wash in
the hybridization reaction is reduced accordingly (for example, if sequences
having > 95%
identity with the probe are sought, the final wash temperature is decreased by
5 C). In practice,
the change in Tm can be between 0.5 C and 1.5 C per 1% mismatch.
Examples of inventive oligonucleotides of length X (in nucleotides), as
indicated by
polynucleotide positions with reference to, e.g., SEQ ID NO:1, include those
corresponding to
sets (sense and antisense sets) of consecutively overlapping oligonucleotides
of length X. where
the oligonucleotides within each consecutively overlapping set (corresponding
to a given X
value) are defined as the finite set of Z oligonucleotides from nucleotide
positions:
n to (n f (X-1));
where n=i, 2, 3,...(Y-(X-1));
where Y equals the length (nucleotides or base pairs) of SEQ ID NO:1(2,280);
where X equals the common length (in nucleotides) of each oligonucleotide in
the set
(e.g., X=20 for a set of consecutively overlapping 20-mers); and
where the number (Z) of consecutively overlapping oligomers of length X for a
given
SEQ ID NO of length Y is equal to Y-(X-1). For example Z= 2,280-19= 2,261 for
either sense
or antisense sets of SEQ ID NO:1, where X=20.
Preferably, the set is limited to those oligomers that comprise at least one
CpQ TpG or.
CpA dinucleotide.
Examples of inventive 20-mer oligonucleotides include the following set of
2,261
oligomers (and the antisense set complementary thereto), indicated by
polynucleotide positions
with reference to SEQ ID NO:1:
1-20, 2-21, 3-22, 4-23, 5-24, ...... 2259-2278, 2260-2279 and 2261-2280.
Preferably, the set is limited to those oligomers that comprise at least one
CpC; TpG or
CpA dinucleotide. '
Likewise, examples of inventive 25-mer oligonucleotides include the following
set of
2,256 oligomers (and the antisense set complementary thereto), indicated by
polynucleotide
positions with reference to SEQ ID NO: 1:
1-25, 2-26, 3-27, 4-28, 5-29, ......2254-2278, 2255-2279 and 2256-2280.
.......... ... .... .
CA 02528853 2005-12-23
Preferably, the set is limited to those oligomers that comprise at least one
CpQ TpG or
CpA dinucleotide.
The present invention encompasses, for each of SEQ ID NOS:l to SEQ ID NO:535
(sense and antisense), multiple consecutively overlapping sets of
oligonucleotides or modified
oligonucleotides of length X, where, e.g., X= 9, 10, 17, 20, 22, 23, 25, 27,
30 or 35 nucleotides.
The oligonucleotides or oligomers according to the present invention
constitute effective
tools useful to ascertain genetic and epigenetic parameters of the genomie
sequence
corresponding to SEQ II) NOS:1 to SEQ ID NO:64. Preferred sets of such
oligonucleotides or
modified oligonucleotides of length X are those consecutively overlapping sets
of oligomers
corresponding to SEQ ID NOS:1 to SEQ ID NO:535 (and to the complements
thereof).
Preferably, said oligomers comprise at least one CpC; TpG or CpA dinucleotide.
Particularly preferred oligonucleotides or oligomers according to the present
invention
are those in which the cytosine of the CpG dinucleotide (or of the
corresponding converted TpG
or CpA dinculeotide) sequences is within the middle third of the
oligonucleotide; that is, where
the oligonucleotide is, for example, 13 bases in length, the CpG, TpG or CpA
dinucleotide is
positioned within the fifth to ninth nucleotide from the 5'-end.
The o ligonucleotides o f the i nvention c an also b e m odified b y c
hemicallly linidng t he
oligonucleotide to one or more moieties or conjugates to enhance the activity,
stability or
detection of the oligonucleotide. Such moieties or conjugates include
chromophores,
fluorophors, lipids such as cholesterol, eholic acid, thioether, aliphatic
c"hains, phospholipids,
polyamines, polyethylene glycol (PEG), palmityl moieties, and others as
disclosed in, for
example, United States Patent Nurnbers 5,514,758, 5,565,552, 5,567,810,
5,574,142, 5,585,481,
5,587,371, 5,597,696 and 5,958,773. The probes may also exist in the form of a
PNA (peptide
nucleic acid) which has particularly p refen-ed pairing properties. Thus, the
oligonucleotide
may include other appended groups such as peptides, and may include
hybridization-triggered
cleavage agents (IK.zol et al., BioTechnfques 6:958-976, 1988) or
intercalating agents (Zon,
Pharm. Res. 5:539-549, 1988). To this end, the oligonucleotide may be
conjugated to another
molecule, e.g., a chromophore, fluorophor, peptide, hybridization-triggered
cross-linldiig agent,
transport agent, hybridization-triggered cleavage agent, etc.
36
CA 02528853 2005-12-23
The oligonucleotide may also comrprix at kast one art recognized modified
sugar and/or
base moiety, or may comprise a modified backbone or non-natural intemucleoside
linkage.
The oligonucleotides or o ligomers according to p articular e mbodiments of
the present
invention are typically used in 'sets,' which contain at least one oligomer
for analysis of each of
the CpG dinucleotides of genomic sequences SEQ ID NOS:1 to SEQ ID NO:64 and
sequences
complementary, thereto, or to the corresponding CpCy, TpG or CpA d'uaucleotide
within a
sequence of the treated nucleic acids accordmg to SEQ ID NOS:304 to SEQ ID
NO:535 and
sequences complementary thereto. However, it is anticipated that for economic
or other factors
it may be preferable to analyze a limited selection of the CpG dinucleofides
within said
sequences, and the content of the set of oligonucleotides is altered
accordingly.
Therefore, in particular embodiments, the present invention provides a set of
at least two
(2) (oligonucleotides and/or PNA-oligomers) useful for detecting the cytosine
methylation stats
in treated genomic DNA (SEQ II) NOS:304 to SEQ ID NO:535), or in genomic DNA
(SEQ ID
NOS:1 to SEQ ID NO:64 and sequences complementary thereto). These probes
enable
diagnosis, classification and/or therapy of genetic and epigenetic parameters
of colorectal cell
proliferative disorders. The sd of oligomers may also be used for detecting
single nucleotide
polymorphisms (SNPs) in treated genomic DNA (SEQ ID NOS:304 to SEQ ID NO:535),
or in.
genomic DNA (SEQ ID NOS:1 to SEQ ID NO:64 and sequences complementary
thereto).
In prefen~ed embodiments, at least one, and more preferably all members of a
set of
oligonucleotides is bound to a solid phase.
ln further embodiments, the present invention provides a set of at least two
(2)
oligonucleotides that are used as 'primer' oligonucleotides for amplifying DNA
sequences of
one of SEQ ID NOS:1 to SEQ ID NO:535 and sequences complementary thereto, or
segments
thereof
It is anticipated that the oligonucleotides may constitute all or part of an
"array" or
'I)NA chip" (i.e., an arrangement of different oligonucleotides and/or PNA-
oligomers bound to
a solid phase). Such an aray of different oligonucleotide- and/or PNA-oligomer
sequences can
be characterized, for example, in that it is arranged on the solid phase in
the form of a
rectangular or hexagonal lattice. The solid-phase surface may be composed of
silicon, glass,
37
CA 02528853 2005-12-23
polystyrene, aluminuniõ steel, iron, copper, nickel, silver, or gold.
Nitrocellulose as well as
plastics such as nylon, which can exist in the fotm of pellets or also as
resin matrices, may also
be used. An overview of the Prior Art in oligomer array manufacturing can be
gathered from a
special edition of Nature Genetics (Nature Genetics Supplement, Volume 21,
January 1999, and
from the literature cited therein). Fluorescently labeled probes are often
used for the scanning of
immobilized DNA arrays. T he s imple attachment o f C y3 a nd C y5 d yes to
the 5 '-OH o f the
specific probe are particularly suitable for fluorescence labels. The
detection of the fluorescence
of the hybridized probes may be carried out, for example, via a confocal
microscope. Cy3 and
Cy5 dyes, besides m.any others, are commercially available.
It is also anticipated that the oligonucleotides, or particular sequences
thereof, may
constitute all or part of an "virtual array" wherein the oligonucleotides, or
particular sequences
thereof, are used, for example, as 'specifiers' as part ot or in combination
with a diverse
population of unique labeled probes to analyze a complex mixture of analytes.
Such a method,
for example is descnbed in US 2003/0013091 (United States serial number
09/898,743,
published 16 January 2003). In such methods, enough labels are generated so
that each nucleic
acid in the complex mixture (i.e., each analyte) can be uniquely bound by a
unique label and
thus detected (each label is directly counted, resulting in a digital read-out
of each molecular
species in the mixture).
It is particularly preferred that the oligomers according to the invention are
utilised for at
least one of detection of; detection and differentiation between or among
subclasses of;
diagnosis of; prognosis of; treatment of; monitoring of; and treatment and
monitoring of
colorectal cell proliferative disorders. This is enabled by use of said sets
for the detection or
detection and differentiation of one or more of the following classes of
tissues: colorectal
carcinoma, colon adenoma, inflammatory colon tissue, grade 2 dysplasia colon
adenomas less
than 1 cm, grade 3 dysplasia colon adenomas larger than 1 cm, normal colon
tissue, non-colon
healthy tissue and non-colon cancer tissue.
Particularly preferrred are those sets of oligomer that comprise at least two
oligonucleotides selected from one of the following sets of oligonucleotides:
SEQ ID NOS:89-285, 14424 - 14453;
38
CA 02528853 2005-12-23
SEQ ID NOS:89 -109,113 - 223, 227 - 293,14424 - 14453;
SEQ ID NOS:89-109, 113-161, 164223, 227-285, 287-293, 14424 - 14453;
SEQ ID NOS:89, 90,126-135,147-151, 224-226, 253-256, 261-267, 283-285;
SEQ ID NOS:89-161, 164-293, 14424 - 14453;
SEQ ID NOS:89-109,113-299,14424 - 14453;
SEQ ID NOS:89-109,113-293, 296-299, 14424 - 14453;
SEQ.ID NOS:1-12, 15-20, 22, 25-36, 38-49, 51-58;
SEQ ID NOS:681 - 683, 683, 684, 684, 685, 685, 686, 686, 687, 687, 688, 688 -
691,
691, 692, 692 - 695, 695, 696, 696 - 699, 699, 700, 700 - 709, 709, 710, 710 -
725, 725, 726,
726, 727, 727, 728, 728 - 760, 760, 761, 761, 762, 762, 763, 763 - 777, 784,
784, 785, 785, 786,
786, 787, 787 - 802, 802, 803, 803 - 814, 814, 815, 815 - 832, 832, 833, 833 -
836, 836, 837,
837 - 872, 872, 873, 873 - 876, 876, 877, 877, 878, 878, 879, 879 - 882, 882,
883, 883 - 892,
892, 893, 893 - 896, 896, 897, 897 - 909, 909, 910, 910, 911, 911, 912, 912 -
915, 915, 916, 916
- 921, 921 - 925, 925, 926, 926 - 939, 939, 940, 940, 941, 941, 942, 942 -
944, 944 - 957, 957,
958, 958, 959, 959, 960, 960, 961, 961, 962, 962 - 971, 971, 972, 972, 973,
973, 974, 974, 975,
975, 976, 976 - 979, 979, 980, 980, 981, 981 - 988, 988, 989, 989 - 994, 994,
995, 995 - 998,
998, 999, 999, 1000, 1000, 1001, 1001, 1002, 1002, 1003, 1003, 1010 - 1016,
1016, 1017, 1017,
1018, 1018, 1019, 1019 - 1028, 1028, 1029, 1029 - 1034, 1034, 1035, 1035 -
1046, 1046, 1047,
1047 - 1058, 1058, 1059, 1059, 1060, 1060, 1061, 1061, 1062, 1062, 1063, 1063 -
1070, 1070,
1071, 1071, 1072, 1072, 1073, 1073, 1074, 1074, 1075, 1075 - 1078, 1078, 1079,
1079 - 1084,
1084, 1085, 1085, 1086, 1086, 1087, 1087 - 1092, 1092, 1093, 1093, 1094, 1094,
1095, 1095 -
1102, 1102, 1103, 1103 - 1114, 1114, 1115, 1115 - 1119, 1119, 1120, 1120 -
1141, 1141, 1142,
1142;
SEQ ID NOS:681 - 683, 683, 684, 684, 685, 685, 686, 686, 687, 687, 688, 688 -
691,
691, 692, 692 - 695, 695, 696, 696 - 699, 699, 700, 700 - 709, 709, 710, 710 -
725, 725, 726,
726, 727, 727, 728, 728 - 760, 760, 761, 761, 762, 762, 763, 763 - 777, 784,
784, 785, 785, 786,
786, 787, 787 - 802, 802, 803, 803 - 814, 814, 815, 815 - 832, 832, 833, 833 -
836, 836, 837,
837 - 872, 872, 873, 873 - 876, 876, 877, 877, 878, 878, 879, 879 - 882, 882,
883, 883 - 885,
39
CA 02528853 2005-12-23
890 - 892, 892, 893, 893 - 896, 896, 897, 897 - 909, 909, 910, 910, 911, 911,
912, 912 - 915,
915, 916, 916 - 921, 921 - 925, 925, 926, 926 - 939, 939, 940, 940, 941, 941,
942, 942 - 944,
944 - 957, 957, 958, 958, 959, 959, 960, 960, 961, 961, 962, 962 - 971, 971,
972, 972, 973, 973,
974, 974, 975, 975, 976, 976 - 979, 979, 980, 980, 981, 981- 988, 988, 989,
989 - 994, 994, 995,
995 - 998, 998, 999, 999, 1000, 1000, 1001, 1001, 1002, 1002, 1003, 1003, 1010
- 1016, 1016,
1017, 1017, 1018, 1018, 1019, 1019 - 1028, 1028, 1029, 1029 - 1034, 1034,
1035, 1035 - 1046,
1046, 1047, 1047 - 1058, 1058, 1059, 1059, 1060, 1060, 1061, 1061, 1062, 1062,
1063, 1063 -
1070, 1070, 1071, 1071, 1072, 1072, 1073, 1073, 1074, 1074, 1075, 1075 - 1078,
1078, 1079,
1079 - 1084, 1084, 1085, 1085, 1086, 1086, 1087, 1087 - 1092, 1092, 1093,
1093, 1094, 1094,
1095,1095-1102,1102,1103,1103-1114,1114,1115,1115-1119,1119,1120,1120-1124,
1129 -1141,1141, 1142,1142;
SEQ ID NOS:738 - 740, 810 - 814, 814, 815, 815 - 829, 854 - 865, 1004 - 1006,
1006,
1007, 1007 - 1009, 1062, 1062, 1063, 1063 - 1069, 1078, 1078, 1079, 1079 -
1084, 1084, 1085,
1085, 1086, 1086, 1087, 1087 -1091,1121-1124;
SEQ ID NOS:681 - 683, 683, 684, 684, 685, 685, 686, 686, 687, 687, 688, 688 -
691,
691, 692, 692 - 695, 695, 696, 696 - 699, 699, 700, 700 - 709, 709, 710, 710 -
725, 725, 726,
726, 727, 727, 728, 728 - 760, 760, 761, 761, 762, 762, 763, 763 - 784, 784,
785, 785, 786, 786,
787, 787 - 802, 802, 803, 803 - 814, 814, 815, 815 - 832, 832, 833, 833 - 836,
836, 837, 837 -
872, 872, 873, 873 - 876, 876, 877, 877, 878, 878, 879, 879 - 882, 882, 883,
883 - 892, 892, 893,
893 - 896, 896, 897, 897 - 909, 909, 910, 910, 911, 911, 912, 912 - 915, 915,
916, 916 - 921,
921 - 925, 925, 926, 926 - 939, 939, 940, 940, 941, 941, 942, 942 - 944, 944 -
957, 957, 958,
958, 959, 959, 960, 960, 961, 961, 962,962 - 971, 971, 972, 972, 973, 973,
974, 974, 975, 975,
976, 976 - 979, 979, 980, 980, 981, 981 - 988, 988, 989, 989 - 994, 994, 995,
995 - 998, 998,
999, 999, 1000, 1000, 1001, 1001, 1002, 1002, 1003, 1003 - 1006, 1006, 1007,
1007 - 1016,
1016, 1017, 1017, 1018, 1018, 1019, 1019 - 1028, 1028, 1029, 1029 - 1034,
1034, 1035, 1035 -
1046, 1046, 1047, 1047 - 1058, 1058, 1059, 1059, 1060, 1060, 1061, 1061, 1062,
1062, 1063,
1063 - 1070, 1070, 1071, 1071, 1072, 1072, 1073, 1073, 1074, 1074, 1075, 1075 -
1078, 1078,
1079, 1079 - 1084, 1084, 1085, 1085, 1086, 1086, 1087, 1087 - 1092, 1092,
1093, 1093, 1094,
1094,1095, 1095 - 1102,1102,1103,1103 - 1114,1114,1115, 1115 -1119, 1119,
1120,1120 -
CA 02528853 2005-12-23
1141, 1141, 1142, 1142 - 1145, 1145, 1146, 1146, 1147, 1147, 1148, 1148 -
1151, 1151, 1152,
1152 - 1154,1154,1155,1155,1156,1156,1157,1157,1158,1158, 1159,1159;
SEQ Il7 NOS:681 - 683, 683, 684, 684, 685, 685, 686, 686, 687, 687, 688, 688 -
691,
691, 692, 692 - 695, 695, 696, 696 - 699, 699, 700, 700 - 709, 709, 710, 710 -
725, 725, 726,
726, 727, 727, 728, 728 - 760, 760, 761, 761, 762, 762, 763, 763 - 784, 784,
785, 785, 786, 786,
787, 787 - 802, 802, 803, 803 - 814, 814, 815, 815 - 832, 832, 833, 833 - 836,
836, 837, 837 -
872, 872, 873, 873 - 876, 876, 877, 877, 878, 878, 879, 879 - 882, 882, 883,
883 - 885, 890 -
892, 892, 893, 893 - 896, 896, 897, 897 - 909, 909, 910, 910, 911, 911, 912,
912 - 915, 915, 916,
916 - 921, 921 - 925, 925, 926, 926 - 939, 939, 940, 940, 941, 941, 942, 942 -
944, 944 - 957,
957, 958, 958, 959, 959, 960, 960, 961, 961, 962, 962 - 971, 971, 972, 972,
973, 973, 974, 974,
975, 975, 976, 976 - 979, 979, 980, 980, 981, 981 - 988, 988, 989, 989 - 994,
994, 995, 995 -
998, 998, 999, 999, 1000, 1000, 1001, 1001, 1002, 1002, 1003, 1003 - 1006,
1006, 1007, 1007 -
1016, 1016, 1017, 1017, 1018, 1018, 1019, 1019 - 1028, 1028, 1029, 1029 -
1034, 1034, 1035,
1035 - 1046, 1046, 1047, 1047 - 1058, 1058, 1059, 1059, 1060, 1060, 1061,
1061, 1062, 1062,
1063, 1063 - 1070, 1070, 1071, 1071, 1072, 1072, 1073, 1073, 1074, 1074, 1075,
1075 - 1078,
1078, 1079, 1079 - 1084, 1084, 1085, 1085, 1086, 1086, 1087, 1087 - 1092,
1092, 1093, 1093,
1094, 1094, 1095, 1095 - 1102, 1102, 1103, 1103 - 1114, 1114, 1115, 1115 -
1119, 1=119, 1120,
1120 - 1141, 1141, 1142, 1142;
SEQ ID NOS:681 - 683, 683, 684, 684, 685, 685, 686, 686, 687, 687, 688, 688 -
691,
691, 692, 692 - 695, 695, 696, 696 - 699, 699, 700, 700 - 709, 709, 710, 710 -
725, 725, 726,
726, 727, 727, 728, 728 - 760, 760, 761, 761, 762, 762, 763, 763 - 777, 784,
784, 785, 785, 786,
786, 787, 787 - 802, 802, 803, 803 - 814, 814, 815, 815 - 832, 832, 833, 833 -
836, 836, 837,
837 - 872, 872, 873, 873 - 876, 876, 877, 877, 878, 878, 879, 879 - 882, 882,
883, 883 - 892,
892, 893, 893 - 896, 896, 897, 897 - 909, 909, 910, 910, 911, 911, 912, 912 -
915, 915, 916, 916
- 921, 921 - 925, 925, 926, 926 - 939, 939, 940, 940, 941, 941, 942, 942 -
944, 944 - 957, 957,
958, 958, 959, 959, 960, 960, 961, 961, 962, 962 - 971, 971, 972, 972, 973,
973, 974, 974, 975,
975, 976, 976 - 979, 979, 980, 980, 981, 981 - 988, 988, 989, 989 - 994, 994,
995, 995 - 998,
998, 999, 999,1000,1000,1001,1001,1002,1002,1003,1003 - 1006,1006,1007,1007 -
1016, 1016, 1017, 1017, 1018, 1018, 1019, 1019 - 1028, 1028, 1029, 1029 -
1034, 1034, 1035,
41
CA 02528853 2005-12-23
1035 - 1046, 1046, 1047, 1047 - 1058, 1058, 1059, 1059, 1060, 1060, 1061,
1061, 1062, 1062,
1063, 1063 - 1070, 1070, 1071, 1071, 1072, 1072, 1073, 1073, 1074, 1074, 1075,
1075 - 1078,
1078, 1079, 1079 - 1084, 1084, 1085, 1085, 1086, 1086, 1087, 1087 -1092, 1092,
1093, 1093,
1094, 1094, 1095, 1095 - 1102, 1102, 1103, 1103 - 1114, 1114, 1115, 1115 -
1119, 1119, 1120,
1120 -1141,1141, 1142, 1142 - 1145, 1145,1146, 1146, 1147,1147, 1148, 1148 -
1151, 1151,
1152, 1152;
and SEQ ID NOS:681 - 683, 683, 684, 684, 685, 685, 686, 686, 687, 687, 688,
688 -
691, 691, 692, 692 - 695, 695, 696, 696 - 699, 699, 700, 700 - 709, 709, 710,
710 - 725, 725,
726, 726, 727, 727, 728, 728 - 760, 760, 761, 761, 762, 762, 763, 763 - 777,
784, 784, 785, 785,
786, 786, 787, 787 - 802, 802, 803, 803 - 814, 814, 815, 815 - 832, 832, 833,
833 - 836, 836,
837, 837 - 872, 872, 873, 873 - 876, 876, 877, 877, 878, 878, 879, 879 - 882,
882, 883, 883 -
892, 892, 893, 893 - 896, 896, 897, 897 - 909, 909, 910, 910, 911, 911, 912,
912 - 915, 915, 916,
916 - 921, 921 - 925, 925, 926, 926 - 939, 939, 940, 940, 941, 941, 942, 942 -
944, 944 - 957,
957, 958, 958, 959, 959, 960, 960, 961, 961, 962, 962 - 971, 971, 972, 972,
973, 973, 974, 974,
975, 975, 976, 976 - 979, 979, 980, 980, 981, 981 - 988, 988, 989, 989 - 994,
994, 995, 995 -
998, 998, 999, 999, 1000, 1000, 1001, 1001,1002, 1002, 1003, 1003 -
1006,1006,1007,1007 -
1016, 1016, 1017, 1017, 1018, 1018, 1019, 1019 - 1028, 1028, 1029, 1029 -
1034, 1034, 1035,
1035 - 1046, 1046, 1047, 1047 - 1058, 1058, 1059, 1059, 1060, 1060, 1061,
1061, 1062, 1062,
1063, 1063 - 1070, 1070, 1071, 1071, 1072, 1072, 1073, 1073, 1074, 1074, 1075,
1075 - 1078,
1078, 1079, 1079 - 1084, 1084, 1085, 1085, 1086, 1086, 1087, 1087 - 1092,
1092, 1093, 1093,
1094, 1094, 1095, 1095 - 1102, 1102, 1103, 1103 -1114, 1114, 1115, 1115 -
1119, 1119, 1120,
1120 - 1141, 1141, 1142, 1142, 1145, 1145, 1146, 1146, 1147, 1147, 1148, 1148 -
1151, 1151,
1152,1152.
In one embodiment of the method, at least one of colorectal carcinoma tissue
or colon
adenomas is distinguished from at least one tissue selected from the group
consisting of
inflaanmatory colon tissue and normal colon tissue. This is achieved by
analysis of the
methylation statns of at least one target sequence comprising, or hybridizing
under stringent
conditions to at least 16 contiguous nucleotides of a sequence selected from
the group consisting
SEQ II} NOS:1-12, 15-20, 22, 25-36. 38-49, 51-58 and complements thereof. This
is
42
CA 02528853 2005-12-23
preferrably achieved by use of a set consisting of at least one
oligonucleotide, and more
preferably at least two selected from one of the groups consisting of, or by
use of a set
comprising of at least two oligonucleotides selected from one of the groups
consisting of:
SEQ ID NOS:89-285 & 14424 - 14453; and
SEQ ID NOS:681 - 683, 683, 684, 684, 685, 685, 686, 686, 687, 687, 688, 688 -
691,
691, 692, 692 - 695, 695, 696, 696 - 699, 699, 700, 700 - 709, 709, 710, 710 -
725, 725, 726,
726, 727, 727, 728, 728 - 760, 760, 761, 761, 762, 762, 763,763 - 784, 784,
785, 785, 786, 786,
787, 787 - 802, 802, 803, 803 - 814, 814, 815, 815 - 832, 832, 833, 833 - 836,
836, 837, 837 -
872, 872, 873,973 - 876, 876, 877, 877, 878, 878, 879, 879 - 882, 882, 883,
883 - 892, 892, 893,
893 - 896, 896, 897, 897 - 909, 909, 910, 910, 911, 911, 912, 912 - 915, 915,
916, 916 - 921,
921 - 925, 925, 926, 926 - 939, 939, 940, 940, 941, 941, 942, 942 - 944, 944 -
957, 957, 958,
958, 959, 959, 960, 960, 961, 961, 962, 962 - 971, 971, 972, 972, 973, 973,
974, 974, 975, 975,
976, 976 - 979, 979, 980, 980, 981, 981 - 988, 988, 989, 989 - 994, 994, 995,
995 - 998, 998,
999, 999, 1000, 1000, 1001, 1001, 1002, 1002, 1003, 1003 - 1006, 1006, 1007,
1007 - 1016,
1016, 1017, 1017, 1018, 1018, 1019, 1019 - 1028, 1028, 1029, 1029 - 1034,
1034, 1035, 1035 -
1046, 1046, 1047, 1047 - 1058, 1058, 1059, 1059, 1060, 1060, 1061, 1061, 1062,
1062, 1063,
1063 - 1070, 1070, 1071, 1071, 1072, 1072, 1073, 1073, 1074, 1074, 1075, 1075 -
1078, 1078,
1079, 1079 - 1084, 1084, 1085, 1085, 1086, 1086, 1087, 1087 - 1092, 1092,
1093, 1093, 1094,
1094,1095, 1095 - 1102, 1102, 1103, 1103 - 1114,1114,1115, 1115 - 1119, 1119,
1120, 1120 -
1124.
In one embodiment of the rnethod, colorectal carcinoma is distinguished from
at least
one tissue selected from the group consisting of non-colon healthy tissue,
periphet~ai blood
lymphocytes and non-colon cancer. In one embodiment this is achieved by
analysis of the
methylation status of at least one target sequence comprising, or hybridizing
under stringent
conditions to at least 16 contiguous nucleotides of a sequence selected from
the group consisting
SEQ II) NOS:1-23, 26-36, 38-43, 45-49, 51-58. This is preferrably achieved by
use of a set
consisting of at least one oligonacleotide, and more preferably at least two
oligonucleotides
selected from one of the groups eonsisting of
SEQ ID NOS:89 - 109,113 - 223, 227 - 293, 14424-14453 ; and
43
CA 02528853 2005-12-23
SEQ ID NOS:681 - 683, 683, 684, 684, 685, 685, 686, 686, 687, 687, 688, 688 -
691,
691, 692, 692 - 695, 695, 696, 696 - 699, 699, 700, 700 - 709, 709, 710, 710 -
725, 725, 726,
726, 727, 727, 728, 728 - 760, 760, 761, 761, 762, 762, 763, 763 - 777, 784,
784, 785, 785, 786,
786, 787, 787 - 802, 802, 803, 803 - 814, 814, 815, 815 - 832, 832, 833, 833 -
836, 836, 837,
837 - 872, 872, 873, 873 - 876, 876, 877, 877, 878, 878, 879, 879 - 882, 882,
883, 883 - 892,
892, 893, 893 - 896, 896, 897, 897 - 909, 909, 910, 910, 911, 911, 912, 912 -
915, 915, 916, 916
- 921, 921 - 925, 925, 926, 926 - 939, 939, 940, 940, 941, 941, 942, 942 -
944, 944 - 957, 957,
958, 958, 959, 959, 960, 960, 961, 961, 962, 962 - 971, 971, 972, 972, 973,
973, 974, 974, 975,
975, 976, 976 - 979, 979, 980, 980, 981, 981 - 988, 988, 989, 999 - 994, 994,
995, 995 - 998,
998, 999, 999, 1000, 1000, 1001, 1001, 1002, 1002, 1003, 1003, 1010 - 1016,
1016, 1017,1017,
1018, 1018, 1019, 1019 - 1028, 1028, 1029, 1029 - 1034, 1034, 1035, 1035 -
1046, 1046, 1047,
1047 - 1058, 1058, 1059, 1059, 1060, 1060, 1061, 1061, 1062, 1062, 1063, 1063 -
1070, 1070,
1071, 1071, 1072, 1072, 1073, 1073, 1074, 1074, 1075, 1075 - 1078, 1078, 1079,
1079 - 1084,
1084, 1085, 1085, 1086, 1086, 1087, 1087 - 1092, 1092, 1093, 1093, 1094, 1094,
1095, 1095 -
1102, 1102, 1103, 1103 - 1114, 1114, 1115, 1115 - 1119, 1119, 1120, 1120 -
1141, 1141, 1142,
1142.
In one embodiment of the method, colorectal carcinoma is distinguished from at
least
one tissue selected from the group consisting of inflammatory colon tissue,
normal colon tissue,
non-colon healthy tissue, peripheral blood lymphocytes, colon adenomas and non-
colon cancer
tissue. This is achieved by analysis of the methylation status of at least one
target sequence
comprising, or hybridizing under stringent conditions to at least 16
contiguous nucleotides of a
sequence selected from the group consisting SEQ ID NO: 1-3, 5-13, 15-23, 26-
36, 38-43, 45-
49, 51-58 and complements thereof. This is preferably achieved by use of a set
consisting of at
least one oligonucleotide, and more preferably at least two by use of a set of
oligomers
comprising at least two oligonucleotides selected from one of the groups
consisting of:
SEQ ID NOS:89 -109,113 - 161, 164 - 223,227 - 285,287 - 293, 14424-14453; and
SEQ ID NOS:681 - 683, 683, 684, 684, 685, 685, 686, 686, 687, 687, 688, 688 -
691,
691, 692, 692 - 695, 695, 696, 696 - 699, 699, 700, 700 - 709, 709, 710, 710 -
725, 725, 726,
726, 727, 727, 728, 728 - 760, 760, 761, 761, 762, 762, 763, 763 - 777, 784,
784, 785, 785, 786,
44
CA 02528853 2005-12-23
786, 787, 787 - 802, 802, 803, 803 - 814, 814, 815, 815 - 832, 832, 833, 833 -
836, 836, 837,
837 - 872, 872, 873, 873 - 876, 876, 877, 877, 878, 878, 879, 879 - 882, 882,
883, 883 - 885,
890 - 892, 892, 893, 893 - 896, 896, 897, 897 - 909, 909, 910, 910, 911, 911,
912, 912 - 915,
915, 916, 916 - 921, 921 - 925, 925, 926, 926 - 939, 939, 940, 940, 941, 941,
942, 942 - 944,
944 - 957, 957, 958, 958, 959, 959, 960, 960, 961, 961, 962, 962 - 971, 971,
972, 972, 973, 973,
974, 974, 975, 975, 976, 976 - 979, 979, 980, 980, 981, 981 - 988, 988, 989,
989 - 994, 994, 995,
995 - 998, 998, 999, 999, 1000, 1000, 1001, 1001, 1002, 1002, 1003, 1003, 1010
- 1016, 1016,
1017, 1017, 1018, 1018, 1019, 1019 - 1028, 1028, 1029, 1029 - 1034, 1034,
1035, 1035 - 1046,
1046, 1047, 1047 - 1058, 1058, 1059, 1059, 1060, 1060, 1061, 1061, 1062, 1062,
1063, 1063 -
1070, 1070, 1071, 1071, 1072, 1072, 1073, 1073, 1074, 1074, 1075, 1075 - 1078,
1078, 1079,
1079 - 1084, 1084, 1085, 1085, 1086, 1086, 1087, 1087 - 1092, 1092, 1093,
1093, 1094, 1094,
1095, 1095 -1102, 1102, 1103, 1103 -1114,1114,1115, 1115 - 1119, 1119, 1120,
1120 - 1124,
1129 - 1141, 1141, 1142, 1142.
In one embodiment of the method, the colorectal carcinoma is distinguished
from colon
adenomas. This is achieved by analysis of the methylation status of at least
one target sequence
comprising, or hybridizing under stringent conditions to at least 16
contiguous nucleotides of a
sequence selected from the group consisting SEQ lI? NO: 11, 25, 27, 38, 40,
45, 53 and '
complements thereot This is prefernrably achieved by use of a set consisting
of at least one
oligonucleotide, and more preferably at least two oligonucleotides selected
from one of the
groups consisting of:
SEQ ID NOS:89, 90, 126 - 135, 147 - 151, 224 - 226, 253 - 256, 261 - 267, 283 -
285;
and
SEQ ID NOS:738 - 740, 810 - 814, 814, 815, 815 - 829, 854 - 865, 1004 - 1006,
1006,
1007, 1007 - 1009, 1062, 1062, 1063, 1063 - 1069, 1078, 1078, 1079, 1079 -
1084, 1084, 1085,
1085, 1086, 1086, 1087, 1087 - 1091, 1121 - 1124.
In one embodiment of the method, at least one of colorectal carcinoma tissue,
or colon
adenomas is distinguished from at least one tissue selected from the group
consisting of
inflammatory colon tissue and normal colon tissue. This is achieved by
analysis of the
methylation status of at least one target sequence comprising, or hybridizing
under stringent
CA 02528853 2005-12-23
conditions to at least 16 contiguous nucleotides of a sequence selected from
the group consisting
SEQ ID NO: 1-64 and complements thereof. This is preferably achieved by use of
a set
consisting of at least one oligonucleotide, and more preferably at least two
oligomers comprising
at least two oligonucleotides selected from one of the groups consisting of:
SEQ ID NOS: 89 - 303,14424-14453; and
SEQ ID NOS:681 - 683, 683, 684, 684, 685, 685, 686, 686, 687, 687, 688, 688 -
691,
691, 692, 692 - 695, 695, 696, 696 - 699, 699, 700, 700 - 709, 709, 710, 710 -
725, 725, 726,
726, 727, 727, 728, 728 - 760, 760, 761, 761, 762, 762, 763, 763 - 784, 784,
785, 785, 786, 786,
787, 787 - 802, 802, 803, 803 - 814, 814, 815, 815 - 832, 832, 833, 833 - 836,
836, 837, 837 -
872, 872, 873, 873 - 876, 876, 877, 877, 878, 878, 879, 879 - 882, 882, 883,
883 - 892, 892, 893,
893 - 896, 896, 897, 897 - 909, 909, 910, 910, 911, 911, 912, 912 - 915, 915,
916, 916 - 921,
921 - 925, 925, 926, 926 - 939, 939, 940, 940, 941, 941, 942, 942 - 944, 944 -
957, 957, 958,
958, 959, 959, 960, 960, 961, 961, 962, 962 - 971, 971, 972, 972, 973, 973,
974, 974, 975, 975,
976, 976 - 979, 979, 980, 980, 981, 981 - 988, 988, 989, 989 - 994, 994, 995,
995 - 998, 998,
999, 999, 1000, 1000, 1001, 1001, 1002, 1002, 1003, 1003 - 1006, 1006, 1007,
1007 - 1016,
1016, 1017, 1017, 1018, 1018, 1019, 1019 - 1028, 1028, 1029, 1029 - 1034,
1034, 1035, 1035 -
1046, 1046, 1047, 1047 - 1058, 1058, 1059, 1059, 1060, 1060, 1061, 1061, 1062,
1062, 1063,
1063 - 1070, 1070, 1071, 1071, 1072, 1072, 1073, 1073, 1074, 1074, 1075, 1075 -
1078, 1078,
1079, 1079 - 1084, 1084, 1085, 1085, 1086, 1086, 1087, 1087 - 1092, 1092,
1093, 1093, 1094,
1094, 1095, 1095 -1102, 1102, 1103, 1103 -1114,1114, 1115, 1115 - 1119, 1119,
1120, 1120 -
1141, 1141, 1142, 1142 - 1145, 1145, 1146, 1146, 1147, 1147, 1148, 1148 -
1151, 115.1, 1152,
1152 -1154, 1154, 1155, 1155, 1156, 1156,1157, 1157, 1158, 1158, 1159, 1159.
In one embodiment of the method, colorectal carcinoma tissue is distingu.ished
from at
least one of inflammatory colon tissue and normal colon tissue. This is
achieved by analysis of
the methylation status of at least one target sequence comprising, or
hybridizing under stringent
conditions to at least 16 contiguous nucleotides of a sequence selected from
the group consisting
SEQ 11) NOS: 1-3, 5-23, 25-36, 38-49, 51-58 and complements thereo~ This is
preferably
achieved by use of a set consisting of at least one oligonucleotide, and more
preferably at least
two oligomers comprising at least two oligonucleotides selected from one of
the groups
46
CA 02528853 2005-12-23
consisting of
SEQ ID NOS: 89 -161,164 - 293, 14424-14453; and
SEQ ID NOS:681 - 683, 683, 684, 684, 685, 685, 686, 686, 687, 687, 688, 688 -
691,
691, 692, 692 - 695, 695, 696, 696 - 699, 699, 700, 700 - 709, 709, 710, 710 -
725, 725, 726,
726, 727, 727, 728, 728 - 760, 760, 761, 761, 762, 762, 763, 763 - 784, 784,
785, 785, 786; 786,
787, 787 - 802, 802, 803, 803 - 814, 814, 815, 815 - 832, 832, 833, 833 - 836,
836, 837, 837 -
872, 872, 873, 873 - 876, 876, 877, 877, 878, 878, 879, 879 - 882, 882, 883,
883 - 885, 890 -
892, 892, 893, 893 - 896, 896, 897, 897 - 909, 909, 910, 910, 911, 911, 912,
912 - 915, 915, 916,
916 - 921, 921 - 925, 925, 926, 926 - 939, 939, 940, 940, 941, 941, 942, 942 -
944, 944 - 957,
957, 958, 958, 959, 959, 960, 960, 961, 961, 962, 962 - 971, 971, 972, 972,
973, 973, 974, 974,
975, 975, 976, 976 - 979, 979, 980, 980, 981, 981 - 988, 988, 989, 989 - 994,
994, 995, 995 -
998, 998, 999, 999, 1000, 1000, 1001, 1001, 1002, 1002, 1003, 1003 - 1006,
1006, 1007, 1007 -
1016, 1016, 1017, 1017, 1018, 1018, 1019, 1019 - 1028, 1028, 1029, 1029 -
1034, 1034, 1035,
1035 - 1046, 1046, 1047, 1047 - 1058, 1058, 1059, 1059, 1060, 1060, 1061,
1061, 1062, 1062,
1063, 1063 - 1070, 1070, 1071, 1071, 1072, 1072, 1073, 10773, 1074, 1074,
1075, 1075 - 1078,
1078, 1079, 1079 - 1084, 1084, 1085, 1085, 1086, 1086, 1087, 1087 - 1092,
1092, 1093, 1093,
1094, 1094, 1095, 1095 - 1102, 1102, 1103, 1103 - 1114, 1114, 1115, 1115 -
1119, 1119, 1120,
1120 - 1141, 1141, 1142, 1142.
In one embodiment of the method, at least one of oolorectal carcinoma tissue,
or colon
adenomas is distinguished from at least one tissue selected from the group
consisting of
inflammatory colon tissue, norrnai colon tissue, non-colon healthy tissue,
peripheral blood
lymphocytes, and non-colon cancer tissue. This is achieved by analysis of the
methylation status
of at least one target sequence comprising, or hybridizing under stringent
conditions to at least
16 contiguous nucleotides of a sequence selected from the group consisting SEQ
ID NO: 1-36,
38-43, 45-58 and complements thereof. This is preferably achieved by use of a
set consisting of
at least one oligonucleotide, and more preferably at least two oligomers
comprising at least two
oligonucleotides selected from one of the groups consisting of:
SEQ IDNOS: 89 -109,113 - 299, 14424-14453; and
SEQ ID NOS:681 - 683, 683, 684, 684, 685, 685, 686, 686, 687, 687, 688, 688 -
691,
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CA 02528853 2005-12-23
In a fiaUier embodiment of the method aerodigestive cell pmliferative
disorders of the
group consisting lung, liver, pancreas, bile duct, stomach, and colon cancers
are detected . Tlais
is achieved by analysis of the methylation status of at least This is achieved
by analysis of the
methylation status of at least one target sequence comprising, or hybridazing
under stringent
conditions to at least 16 contiguous nucleotides of a sequence selected from
the group consisting
SEQ ID NO: 20, 41, 35, 3, 39, 33 and complements thereof.The present invention
further
provides a method for ascertaining genetic and/or epigenetic parameters of the
genomic
sequences according to SEQ ID NOS:1 to SEQ ID NO:64 within a subject by
analyzing cytosine
methylation and single nucleotide polymorphisms. Said rnethod comprising
contacting a nucleic
acid eomprising one or more of SEQ ID NOS:1 to SEQ ID NO:64 in a biological
sample
obtamed from said subject with at least one reagent or a series of reagents,
wherein said reagent
or series of reagents, distinguishes between methylated and non-nnethylated
CpG dinucleotides
within the target nucleic acid.
Preferably, said method comprises the follownng steps: In the first step, a
sample of the
tissue to be analysed is obtained. The source may be any suitable source, sueh
as cell lines,
ba,stological slides, biopsies, paraffin-embedded tissue, bodily fluids,
stool, coloxuc effluent,
urine, b lood p lasma, b lood s eram, w hole b lood, i solated b lood c ells,
c ells i solated from t he
blood and all possible combinations thereof. It is preferred that said
sources. ofDNA are stool or
bodily fluids selected from the group consisting colonic effluent , urine,
blood plasma, blood
serum, whole blood, isolated blood cells, cells isolated from the blood.
The genomic DNA is then isolated from the sample. Genomic DNA may be isolated
by
any means standard in the art, including the use of commercially available
kits. Briefly, wherein
the DNA of interest is encapsulated in by a cellular membrane the biological
sample must be
disrapted and lysed by enzymaiic, chemical or mechanical means. The DNA
solution may then
be cleared of proteins and other contaminants e.g. by digestion with
proteinase K. The genomic
DNA is then reoovered from the solution. This may be carri.ed out by means of
a variety of
methods including salting out, organic extraction or binding of the DNA to a
solid phase
setpport. The choice of method will be affected by several factors inncluding
time, expense and
required quantity of DNA.
CA 02528853 2005-12-23
Once the nucleic acids have been extracted, the genomic double stranded DNA is
used in
the analysis.
In the secoitd step of the method, the genomic DNA sample is treated in such a
manner
that cytosine bases which are unmethylated at the 5'-position are converted to
uracil, thymine, or
another base which is dissimilar to cytosine in terms of hybridization
behavior. This will be
understood as 'pretreatment' or 'treatment' herein.
The above-desennbed treatment of genomic DNA is preferably carried out with
bisulfite
(hydrogen sulfite, disulfite) and subsequent allraline hydrolysis which
results in a conversion of
non~nethylated cytosine nucleobases to uracil or to another base which is
dissimilar to cytosine
in terms of base pairing behavior.
In the third step of the method, fragments of the treated DNA are ampli$ed,
using sets of
primer oligonucleotides according to the present invention, and an
amplification enzyme. The
amplification of several DNA segments can be carried out simultaneously in one
and the same
reaction vessel. Typically, the amplification is carried out using a
polymerase chain reaction
(PCR). The set of primer oligonucleotides includes at least two
oligornucleotides whose
sequences are each reverse complementary, identical, or hybridize under
stringent or highly,
stringent conditions to an at least 16-base-pair long segment of the base
sequences of one.of.
SEQ ID NOS:304 to SEQ ID NO:535 and sequences complementary thereto.
In an altemate e mbodiment o f t he method, the methylation s tatus o
fpreselected CpG
positions within the nucleic acid sequences comprising one or more of SEQ ID
NOS:1 to SEQ
ID NO:64 may be detected by use of methylation-specific primer
oligomicleotides. This
technique (MSP) has been described in United States Patent No. 6,265,171 to
Herman. The use
of inethylation status specific primers for the amplification of bisulfite
treated DNA allows the
differentiation between mettrylated and unmethylated nucleic acids. MSP
primers pairs contain
at least one primer which hybridizes to a bisulfite treated CpG dinucleotide.
Therefore, the
sequence of said primers comprises at least one CpG dinucleotide. MSP primers
specific for
non-methylated DNA contain a"T' at the position of the C position in the CpG.
Preferably,
therefore, the base sequence of said primers is required to comprise a
saquence having a length
of at least 9 nucleotides which hybridizes to a treated nucleic acid sequence
according to one of
51
CA 02528853 2005-12-23
SEQ ID NOS:304 to SEQ ID NO:535 and sequences complementary thereto, wherein
the base
sequence of said oligomers comprises at least one CpG dinucleotide.
In a further prefezred embodiment of the method, the MSP primers are selected
from the
group consisting SEQ ID NOS:2754, 2757, 2759, 2767, 2768, 2772, 2776, 2779,
2784, 2785,
2786, 2789, 2791, 2792, 2794, 2797, 2798, 2801, 2805, 2806, 2809, 2811, 2815,
2817, 2824,
2830, 2832, 2833, 2834, 2838, 2840, 2843, 2846, 2850, 2851, 2853, 2855, 2859,
2866, 2867,
2870, 2871, 2876, 2878, 2881, 2885, 2886, 2892, 2893, 2895, 2896, 2898, 2904,
2912, 2915,
2917, 2919, 2922, 2923, 2929, 2931, 2939, 2941, 2944, 2946, 2948, 2950, 2955,
2956, 2957,
2960, 2962, 2965, 2966, 2967, 2968, 2969, 2971, 2975, 2976, 2977, 2978, 2979,
2980, 2981,
2984, 2988, 2989, 2990, 2992, 2994, 2997, 2999, 3001, 3002, 3004, 3006, 3007,
3008, 3010,
3013, 3014, 3016, 3017, 3018, 3020, 3022, 3025, 2752, 2755, 2760, 2762, 2763,
2764, 2765,
2769, 2771, 2773, 2775, 2777, 2780, 2781, 2782, 2783, 2787, 2788, 2790, 2793,
2795, 2796,
2800, 2802, 2803, 2804, 2808, 2810, 2812, 2814, 2816, 2818, 2820, 2821, 2823,
2825, 2827,
2828, 2829, 2831, 2835, 2836, 2837, 2839, 2841, 2842, 2844, 2845, 2847, 2849,
2852, 2854,
2856, 2858, 2860, 2861, 2862, 2863, 2864, 2865, 2868, 2869, 2872, 2873, 2874,
2875, 2877,
2879, 2880, 2882, 2883, 2884, 2887, 2888, 2889, 2890, 2891, 2894, 2897, 2899,
2901, 2902,
2903, 2905, 2907, 2909, 2911, 2914, 2916, 2918, 2920, 2924, 2926, 2927, 2928,
2930, 2932,
2933, 2934, 2935, 2936, 2937, 2938, 2942, 2945, 2947, 2949, 2951, 2953, 2954,
2958, 2959,
2961, 2963, 2970, 2972, 2973, 2974, 2982, 2985, 2987, 2991, 2993, 2995, 2996,
2998, 3000,
3003, 3005, 3011, 3019, 3021, 3023, 3026, 3027, 3028, 3032, 3033, 3036, 3037,
3038, 3039,
3041, 3042, 3043, 3044, 3047, 3048, 3049, 3052, 3055, 3059, 3061, 3064, 3065,
3068, 3069,
3071, 3072, 3075, 3076, 3080, 3083, 3084, 3085, 3086, 3091, 3093, 3096, 3097,
3100, 3104,
3109, 3110, 3113, 3115, 3117, 3118, 3123, 3126, 3127, 3130, 3134, 3135, 3136,
3138, 3139,
3144, 3146, 3147, 3149, 3150, 3155, 3029, 3031, 3034, 3040, 3045, 3050, 3051,
3053, 3054,
3056, 3057, 3060, 3063, 3066, 3073, 3078, 3081, 3088, 3089, 3090, 3092, 3094,
3098, 3101,
3103, 3105, 3107, 3108, 3111, 3114, 3116, 3119, 3121, 3122, 3124, 3128, 3131,
3133, 3137,
3140, 3142, 3145, 3148, 3151, 3152, 3153, 3157, 3159, 3165, 3166, 3167, 3170,
3171, 3172,
3174, 3175, 3176, 3179, 3180, 3181, 3184, 3185, 3186, 3192, 3194, 3198, 3201,
3202, 3203,
3204, 3206, 3209, 3212, 3213, 3215, 3216, 3217, 3218, 3221, 3224, 3226, 3228,
3229, 3230,
52
CA 02528853 2005-12-23
3232, 3233, 3234, 3235, 3237, 3238, 3239, 3245, 3247, 3248, 3249, 3160, 3162,
3163, 3164,
3168, 3169, 3173, 3178, 3182, 3183, 3187, 3189, 3190, 3195, 3196, 3197, 3199,
3205, 3207,
3210, 3214, 3219, 3222, 3223, 3225, 3227, 3236, 3240, 3242, 3243, 3244, 3246,
3250, 3252,
3256, 3261, 3264, 3268, 3269, 3270, 3273, 3275, 3279, 3280, 3281, 3282, 3283,
3284, 3285,
3286, 3290, 3292; 3295, 3297, 3301, 3304, 3305, 3306, 3307, 3308, 3310, 3311,
3312, 3315,
3319, 3320, 3324, 3327, 3331, 3333, 3334, 3339, 3348, 3351, 3354, 3358, 3360,
3364, 3367,
3368, 3374, 3376, 3253, 3255, 3257, 3259, 3260, 3262, 3265, 3266, 3267, 3271,
3274, 3276,
3278, 3287, 3289, 3291, 3293, 3298, 3300, 3302, 3309, 3313, 3314, 3316, 3318,
3321, 3322,
3325, 3328, 3330, 3332, 3335, 3337, 3338, 3340, 3342, 3343, 3344, 3345, 3346,
3347; 3349,
3350, 3352, 3353, 3355, 3356, 3357, 3359, 3361, 3363, 3365, 3369, 3371, 3372,
3373, 3377,
3380, 3383, 3386, 3388, 3389, 3390, 3391, 3392, 3395, 3396, 3400, 3401, 3403,
3406, 3408,
3413, 3414, 3417, 3420, 3423, 3428, 3432, 3433, 3434, 3436, 3439, 3440, 3442,
3443, 3445,
3447, 3453, 3455, 3458, 3463, 3466, 3469, 3470, 3471, 3472, 3473, 3474, 3475,
3477, 3478,
3480, 3481, 3483, 3484, 3487, 3490, 3494, 3495, 3496, 3497, 3499, 3500, 3504,
3505, 3508,
3512, 3514, 3515, 3516, 3521, 3525, 3529, 3533, 3534, 3537, 3538, 3540, 3541,
3542, 3543,
3544, 3547, 3548, 3553, 3557, 3558, 3559, 3560, 3562, 3563, 3566, 3571, 3574,
3575, 3577, .
3580, 3583, 3584, 3585, 3586, 3592, 3594, 3595, 3597, 3598, 3599, 3601, 3603,
3604, 3607, .
3608, 3612, 3619, 3621, 3622, 3626, 3631, 3632, 3637, 3638, 3639, 3640, 3642,
3643, 3644,
3646, 3647, 3649, 3650, 3652, 3656, 3657, 3660, 3663, 3668, 3670, 3673, 3676,
3677, 3678,
3683, 3688, 3690, 3694, 3697, 3700, 3702, 3703, 3704, 3708, 3404, 3409, 3411,
3415, 3418,
3419, 3421, 3424, 3426, 3427, 3429, 3431, 3435, 3437, 3438, 3441, 3444, 3446,
3448, 3450,
3452, 3454, 3456, 3459, 3461, 3462, 3464, 3467, 3476, 3479, 3482, 3485, 3488,
3489, 3491,
3492, 3493, 3501, 3503, 3506, 3509, 3510, 3511, 3513, 3517, 3518, 3519, 3520,
3522, 3524,
3526, 3527, 3528, 3530, 3531, 3535, 3536, 3545, 3549, 3551, 3552, 3554, 3555,
3556, 3561,
3564, 3565, 3568, 3569, 3570, 3573, 3576, 3578, 3581, 3587, 3588, 3589, 3590,
3591, 3593,
3596, 3600, 3602, 3605, 3606, 3609, 3611, 3613, 3614, 3615, 3616, 3617, 3618,
3623, 3624,
3625, 3627, 3629, 3630, 3633, 3634, 3635, 3636, 3641, 3645, 3651, 3653, 3654,
3655, 3658,
3661, 3662, 3665, 3666, 3667, 3669, 3671, 3674, 3679, 3681, 3684, 3686, 3689,
3691, 3692,
3693, 3695, 3696, 3698, 3701, 3705, 3706, 3707, 3709, 3710, 3714, 3715, 3717,
3718, 3722,
53
CA 02528853 2005-12-23
3723, 3729, 3730, 3732, 3735, 3739, 3740, 3741, 3743, 3744, 3745, 3746, 3749,
3753, 3754,
3755, 3756, 3711, 3713, 3719, 3721, 3724, 3725, 3726, 3727, 3728, 3731, 3733,
3736, 3737,
3738, 3742, 3747, 3748, 3750, 3752, 3757, 3758, 3759, 3760, 3761, 3764, 3767,
3771, 3773,
3775, 3776, 3777, 3781, 3785, 3786, 3787, 3790, 3791, 3793, 3796, 3797, 3798,
3799, 3805,
3809, 3814, 3817, 3818, 3822, 3823, 3828, 3833, 3836, 3844, 3847, 3850, 3851,
3853, 3856,
3857, 3862, 3873, 3876, 3877, 3880, 3885, 3887, 3889, 3890, 3892, 3893, 3895,
3896, 3899,
3902, 3903, 3905, 3906, 3907, 3908, 3916, 3923, 3926, 3927, 3930, 3935, 3936,
3938, 3939,
3940, 3944, 3945, 3949, 3950, 3951, 3954, 3955, 3957, 3960, 3965, 3970, 3971,
3978, 3979,
3982, 3983, 3984, 3985, 3762, 3765, 3768, 3770, 3772, 3774, 3778, 3779, 3780,
3782, 3784,
3788, 3792, 3794, 3800, 3801, 3802, 3803, 3804, 3806, 3807, 3808, 3810, 3812,
3813, 3815,
3819, 3820, 3821, 3824, 3826, 3827, 3829, 3831, 3832, 3834, 3837, 3838, 3839,
3840, 3842,
3845, 3848, 3852, 3854, 3858, 3860, 3861, 3863, 3864, 3865, 3866, 3867, 3868,
3869, 3870,
3871, 3872, 3874, 3875, 3878, 3881, 3883, 3884, 3886, 3888, 3891, 3894, 3897,
3898, 3900,
3901, 3904, 3909, 3411, 3914, 3915, 3917, 3919, 3920, 3921, 3922, 3924, 3928,
3931, 3933,
3934, 3937, 3941, 3942, 3943, 3947, 3948, 3952, 3958, 3959, 3961, 3962, 3963,
3964, 3966,
3968, 3972, 3974, 3975, 3976, 3980, 3981, 3986, 3987, 3990, 3993, 3994, 3997,
4000, 4003,
4004, 4006, 4012, 4015, 4017, 4020, 4024, 4027, 4029, 4031, 4032, 4033, 4037,
4038, 4040,
4042, 4047, 4048, 4050, 4051, 4052, 4054, 4055, 4058, 4063, 4066, 4069, 4070,
4072, 4073,
4076, 4078, 4079, 4082, 4083, 4084, 4085, 4086, 4090, 4093, 4094, 4095, 4096,
4099, 4100,
4104, 4106, 4113, 4116, 4119, 4120, 4124, 4127, 4130, 4134, 4135, 4136, 4138,
4142, 4145,
4146, 4149, 4150, 4152, 4153, 4159, 4160, 4161, 4163, 4168, 4169, 4171, 4173,
4176, 4180,
4181, 4185, 4189, 4191, 4192, 4194, 4197, 4198, 4199, 4201, 4206, 4207, 4208,
4213, 4219,
4220, 4223, 4224, 4225, 4228, 4231, 4232, 4233, 4237, 4238, 4242, 4243, 4246,
4247, 4251,
4254, 4255, 4256, 4258, 3988, 3991, 3995, 3998, 4001, 4005, 4007, 4009, 4010,
4011, 4013,
4016, 4018, 4019, 4021, 4022, 4023, 4025, 4028, 4034, 4035, 4039, 4043, 4045,
4046, 4049,
4056, 4059, 4061, 4064, 4065, 4067, 4068, 4071, 4074, 4075, 4077, 4080, 4087,
4088, 4089,
4092, 4098, 4101, 4102, 4105, 4108, 4109, 4110, 4111, 4112, 4114, 4115, 4117,
4121, 4122,
4123, 4125, 4128, 4129, 4131, 4132, 4133, 4137, 4139, 4141, 4144, 4148, 4151,
4154, 4156,
4157, 4162, 4165, 4166, 4167, 4170, 4172, 4174, 4177, 4179, 4182, 4184, 4186,
4188, 4190,
54
CA 02528853 2005-12-23
4195, 4196, 4202, 4204, 4205, 4209, 4211, 4214, 4216, 4217, 4218, 4221, 4226,
4229, 4234,
4236, 4239, 4241, 4244, 4245, 4248, 4249, 4252, 4257, 4259, 4260, 4261, 4262,
4266, 4267,
4268, 4271, 4272, 4275, 4276, 4279, 4281, 4282, 4284, 4285, 4287, 4289, 4290,
4292, 4295,
4297, 4299, 4301, 4306, 4310, 4316, 4317, 4318, 4321, 4323, 4325, 4328, 4334,
4335, 4337,
4341, 4342, 4343, 4344, 4348, 4350, 4351, 4352, 4354, 4357, 4358, 4359, 4364,
4365, 4366,
4370, 4371, 4372, 4373, 4376, 4378, 4381, 4383, 4384, 4386, 4388, 4396, 4397,
4399, 4400,
4403, 4405, 4407, 4409, 4412, 4415, 4416, 4417, 4424, 4426, 4429, 4433, 4435,
4437, 4442,
4443, 4447, 4450, 4451, 4452, 4455, 4457, 4460, 4461, 4462, 4465, 4466, 4469,
4470, 4474,
4478, 4481, 4484, 4488, 4491, 4494, 4495, 4497, 4498, 4504, 4506, 4509, 4510,
4511, 4514,
4516, 4518, 4520, 4521, 4523, 4525, 4528, 4530, 4533, 4534, 4535, 4538, 4540,
4542, 4545,
4547, 4548, 4553, 4554, 4555, 4556, 4557, 4562, 4564, 4567, 4568, 4569, 4579,
4580, 4582,
4584, 4587, 4589, 4593, 4596, 4597, 4598, 4600, 4601, 4602, 4605, 4606, 4607,
4609, 4610,
4613, 4614, 4616, 4618, 4621, 4624, 4626, 4627, 4628, 4631, 4632, 4636, 4639,
4641, 4644,
4645, 4648, 4263, 4265, 4269, 4270, 4273, 4274, 4277, 4280, 4283, 4286, 4288,
4291, 4293,
4296, 4298, 4300, 4302, 4303, 4304, 4305, 4307, 4308, 4309, 4311, 4313, 4315,
4319, 4322,
4324, 4326, 4327, 4330, 4331, 4332, 4333, 4336, 4338, 4340, 4345, 4347, 4349,
4353, 4355,
4360, 4361, 4362, 4367, 4368, 4369, 4374, 4375, 4377, 4379, 4382, 4385, 4387,
4389, 4390,
4391, 4392, 4393, 4394, 4395, 4398, 4401, 4406, 4408, 4410, 4411, 4413, 4418,
4420, 4422,
4423, 4425, 4427, 4428, 4430, 4432, 4434, 4436, 4438, 4440, 4441, 4444, 4446,
4448, 4453,
4454, 4456, 4458, 4463, 4464, 4467, 4471, 4472, 4473, 4475, 4476, 4477, 4479,
4480, 4482,
4485, 4486, 4487, 4489, 4490, 4492, 4496, 4499, 4501, 4502, 4503, 4505, 4507,
4508, 4512,
4515, 4517, 4519, 4522, 4524, 4529, 4531, 4536, 4537, 4539, 4541, 4543, 4546,
4549, 4551,
4558, 4559, 4560, 4561, 4563, 4565, 4570, 4571, 4572, 4573, 4574, 4575, 4576,
4578, 4581,
4583, 4585, 4586, 4588, 4590, 4592, 4595, 4599, 4603, 4611, 4612, 4615, 4617,
4619, 4620,
4622, 4630, 4633, 4634, 4635, 4638, 4640, 4642, 4643, 4646, 4647, 4649, 4652,
4655, 4659,
4661, 4663, 4664, 4665, 4668, 4671, 4674, 4675, 4676, 4678, 4680, 4683, 4686,
4690, 4696,
4700, 4703, 4704, 4706, 4709, 4711, 4714, 4715, 4717, 4718, 4719, 4720, 4721,
4726, 4727,
4730, 4732, 4738, 4742, 4745, 4749, 4750, 4753, 4755, 4759, 4763, 4768, 4769,
4772, 4773,
4776, 4778, 4779, 4782, 4784, 4789, 4790, 4794, 4797, 4801, 4805, 4807, 4813,
4814, 4819,
CA 02528853 2005-12-23
4821, 4822, 4827, 4829, 4833, 4834, 4835, 4837, 4838, 4841, 4842, 4843, 4844,
4847, 4849,
4850, 4852, 4853, 4859, 4860, 4861, 4865, 4867, 4868, 4869, 4870, 4873, 4876,
4877, 4878,
4879, 4883, 4885, 4889, 4891, 4892, 4894, 4895, 4897, 4898, 4903, 4906, 4911,
4912, 4918,
4919, 4920, 4924, 4927, 4929, 4932, 4934, 4941, 4942, 4945, 4948, 4949, 4950,
4650, 4653,
4656, 4658, 4660, 4662, 4666, 4667, 4669, 4670, 4672, 4677, 4681, 4684, 4685,
4687, 4688,
4689, 4691, 4693, 4694, 4695, 4697, 4699, 4701, 4702, 4705, 4707, 4708, 4716,
4722, 4724,
4728, 4731, 4733, 4735, 4736,4737, 4739, 4741, 4744, 4746, 4748, 4751, 4754,
4756, 4758,
4760, 4761, 4764, 4766, 4767, 4770, 4771, 4774, 4777, 4780, 4783, 4785, 4786,
4787, 4788,
4791, 4792, 4793, 4795, 4798, 4800, 4802, 4804, 4806, 4808, 4810, 4811, 4812,
4815, 4816,
4817, 4818, 4820, 4823, 4824, .4825, 4828, 4830, 4832, 4836, 4839, 4840, 4845,
4846, 4848,
4851, 4854, 4855, 4856, 4857, 4858, 4862, 4864, 4871, 4872, 4874, 4875, 4881,
4882, 4884,
4886, 4887, 4890, 4893, 4896, 4899, 4900, 4901, 4902, 4904, 4905, 4907, 4908,
4909, 4910,
4913, 4915, 4917, 4921, 4922, 4923, 4925, 4926, 4928, 4930, 4935, 4936, 4937,
4938, 4940,
4943, 4944, 4946, 4947, 4951, 4952, 4953, 4954, 4956, 4957, 4960, 4963, 4967,
4970, 4971,
4972, 4976, 4977, 4985, 4986, 4987, 4989, 4991, 4992, 4997, 4999, 5002, 5003,
5004, 5006,
5010, 5013, 5014, 5015, 5017, 5018, 5019, 5020, 5021, 5024, 5025, 5027, 5031,
5032, 5033,
5037, 5040, 5041, 5042, 5043, 4958, 4961, 4964, 4966, 4968, 4969, 4973, 4974,
4975, 4978,
4979, 4980, 4981, 4982, 4983, 4984, 4988, 4990, 4993, 4995, 4996, 4998, 5000,
5005, 5007,
5009, 5011, 5016, 5022, 5026, 5028, 5030, 5034, 5036, 5038, 5039, 5044, 5047,
5049, 5050,
5052, 5054, 5055, 5057, 5060, 5061, 5064, 5045, 5048, 5051, 5053, 5056, 5058,
5062, 5065,
5056, 5069, 5073, 5077, 5080, 5083, 5087, 5093, 5096, 5099, 5100, 5103, 5104,
5106, 5111,
5116, 5119, 5120, 5125, 5128, 5132, 5134, 5135, 5137, 5139, 5140, 5142, 5144,
5148, 5149,
5150, 5155, 5157, 5158, 5160, 5166, 5168, 5170, 5171, 5173, 5176, 5177, 5179,
5184, 5187,
5188, 5190, 5193, S195, 5198, 5201, 5204, 5208, 5211, 5215, 5216, 5217, 5220,
5221, 5222,
5224, 5226, 5229, 5230, 5231, 5232, 5234, 5237, 5242, 5244, 5245, 5246, 5247,
5249, 5250,
5252, 5254, 5255, 5256, 5261, 5262, 5264, 5265, 5273, 5274, 5276, 5278, 5279,
5281, 5284,
5285, 5286, 5287, 5288, 5289, 5292, 5296, 5300, 5303, 5304, 5307, 5308, 5309,
5313, 5315,
5317, 5318, 5322, 5323, 5326, 5329, 5330, 5333, 5336, 5337, 5341, 5343, 5348,
5349, 5352,
5354, 5355, 5358, 5359, 5361, 5362, 5363, 5364, 5365, 5367, 5369, 5370, 5372,
5375, 5377,
56
CA 02528853 2005-12-23
5379, 5382, 5385, 5387, 5393, 5394, 5395, 5398, 5400, 5404, 5407, 5408, 5409,
5412, 5413,
5414, 5416, 5418, 5421, 5424, 5426, 5428, 5431, 5432, 5433, 5434, 5435, 5437,
5440, 5441,
5443, 5448, 5450, 5451, 5454, 5455, 5458, 5460, 5461, 5464, 5468, 5470, 5471,
5474, 5475,
5477, 5482, 5484, 5485, 5487, 5490, 5491, 5492, 5494, 5496, 5498, 5499, 5500,
5501, 5502,
5503, 5504, 5506, 5509, 5510, 5512, 5516, 5517, 5518, 5524, 5525, 5526, 5528,
5531, 5533,
5534, 5535, 5536, 5537, 5540, 5542, 5544, 5545, 5546, 5549, 5550, 5552, 5553,
5559, 5561,
5566, 5569, 5572, 5573, 5575, 5577, 5578, 5579, 5580, 5581, 5582, 5588, 5593,
5595, 5596,
5598, 5600, 5601, 5603, 5604, 5605, 5608, 5614, 5615, 5616, 5619, 5622, 5624,
5625, 5628,
5629, 5633, 5634, 5637, 5638, 5639, 5643, 5644, 5645, 5647, 5648, 5649, 5650,
5654, 5662,
5664, 5067, 5070, 5072, 5074, 5075, 5076, 5078, 5081, 5084, 5086, 5088, 5090,
5091, 5094,
5097, 5101, 5105, 5107, 5109, 5110, 5112, 5114, 5115, 5117, 5121, 5123, 5124,
5126, 5129,
5131, 5136, 5143, 5145, 5147, 5151, 5153, 5156, 5159, 5161, 5162, 5163, 5164,
5165, 5167,
5169, 5172, 5174, 5175, 5180, 5181, 5182, 5183, 5185, 5186, 5189, 5191, 5194,
5196, 5199,
5200, 5202, 5203, 5205, 5207, 5209, 5210, 5212, 5214, 5218, 5219, 5223, 5225,
5227, 5233,
5235, 5238, 5239, 5240, 5241, 5248, 5251, 5253, 5257, 5259, 5260, 5263, 5266,
5268, 5269,
5270, 5271, 5272, 5280, 5282, 5290, 5291, 5293, 5295, 5297, 5299, 5301, 5306,
5310, 5311,
5312, 5314, 5316, 5319, 5321, 5324, 5327, 5331, 5332, 5334, 5338, 5339, 5340,
5342, 5344,
5346, 5347, 5351, 5353, 5356, 5360, 5366, 5368, 5371, 5373, 5376, 5378, 5380,
5383, 5388, .
5389, 5391, 5392, 5396, 5399, 5401, 5403, 5405, 5410, 5415, 5417, 5419, 5420,
5422, 5425,
5427, 5429, 5436, 5438, 5442, 5444, 5445, 5446, 5447, 5449, 5452, 5456, 5457,
5459, 5462,
5465, 5466, 5467, 5469, 5472, 5478, 5480, 5481, 5486, 5488, 5493, 5495, 5497,
5505, 5507,
5508, 5511, 5513, 5515, 5519, 5521, 5522, 5527, 5529, 5532, 5538, 5539, 5541,
5543, 5548,
5551, 5554, 5555, 5556, 5557, 5558, 5560, 5562, 5563, 5564, 5565, 5567, 5568,
5570, 5571,
5574, 5576, 5583, 5585, 5586, 5587, 5589, 5591, 5592, 5594, 5597, 5599, 5602,
5606, 5609,
5611, 5612, 5618, 5620, 5623, 5626, 5627, 5630, 5632, 5635, 5640, 5642, 5646,
5651, 5652,
5656, 5657, 5659, 5660, 5661, 5663, 5665, 5667, 5670, 5672, 5673, 5676, 5678,
5679, 5681,
5682, 5683, 5686, 5687, 5688, 5690, 5694, 5697, 5698, 5699, 5700, 5701, 5704,
5708, 5709,
5711, 5713, 5714, 5717, 5721, 5723, 5724, 5727, 5728, 5733, 5734, 5735, 5736,
5739, 5740,
5741, 5742, 5743, 5668, 5671, 5674, 5677, 5680, 5684, 5689, 5691, 5693, 5696,
5702, 5705,
57
CA 02528853 2005-12-23
5707, 5710, 5712, 5715, 5718, 5720, 5722, 5725, 5729, 5730, 5731, 5732, 5737,
5744, 5747,
5750, 5753, 5759, 5762, 5763, 5770, 5775, 5777, 5778, 5780, 5782, 5785, 5787,
5791, 5792,
5793, 5794, 5797, 5801, 5803, 5808, 5810, 5812, 5814, 5816, 5817, 5824, 5832,
5833, 5834,
5835, 5838, 5840, 5841, 5844, 5849, 5853, 5854, 5857, 5859, 5860, 5862, 5863,
5865, 5868,
5874, 5882,.5883, 5885, 5887, 5894, 5895, 5896, 5897, 5902, 5904, 5906, 5909,
5912, 5915,
5918, 5919, 5920, 5922, 5923, 5926, 5927, 5928, 5930, 5932, 5933, 5934, 5935,
5939, 5940,
5946, 5947, 5948, 5949, 5952, 5954, 5955, 5956, 5958, 5959, 5961, 5962, 5964,
5965, 5968,
5970, 5971, 5972, 5973, 5977, 5978, 5980, 5983, 5986, 5987, 5992, 5993, 5996,
5997, 6000,
6002, 6003., 6004, 6005, 6006, 6008, 6009, 6010, 6011, 6012, 6013, 6017, 6018,
6019, 6020,
6025, 6027, 6030, 6033, 6035, 6036, 6039, 6041, 6042, 6045, 6049, 6050, 6051,
6055, 6059,
6063, 6064, 6065, 6067, 6070, 6072, 6073, 6075, 6077, 6078, 6081, 5745, 5748,
5751, 5754,
5756, 5758, 5760, 5764, 5766, 5767, 5768, 5769, 5771, 5773, 5774, 5776, 5779,
5781, 5783,
5786, 5788, 5789, 5790, 5795, 5796, 5798, 5800, 5802, 5804, 5806, 5807, 5809,
5811, 5813,
5818, 5820, 5821, 5822, 5823, 5825, 5826, 5827, 5828, 5830, 5831, 5836, 5837,
5839, 5842,
5843, 5845, 5846, 5847, 5848, 5850, 5852, 5855, 5856, 5858, 5861, 5864, 5866,
5867, 5870,
5871, 5872, 5873, 5875, 5876, 5877, 5878, 5879, 5880, 5881, 5884, 5886, 5888,
5889, 5890,
5891, 5892, 5893, 5898, 5899, 5900, 5903, 5905, 5907, 5910, 5913, 5916, 5921,
5924, 5929,
5931, 5936, 5937, 5938, 5942, 5943, 5944, 5945, 5950, 5953, 5957, 5960, 5963,
5966, 5969,
5975, 5976, 5981, 5984, 5988, 5990, 5991, 5994, 5998, 5999, 6001, 6007, 6014,
6015, 6016,
6021, 6023, 6024, 6026, 6028, 6031, 6032, 6034, 6037, 6038, 6040, 6043, 6046,
6048, 6053,
6054, 6056, 6058, 6060, 6062, 6066, 6068, 6074, 6076, 6079, 6083, 6084, 6085,
6089, 6090,
6093, 6096, 6098, 6100, 6106, 6110, 6111, 6114, 6118, 6121, 6127, 6130, 6133,
6135, 6137,
6138, 6141, 6143, 6145, 6148, 6150, 6151, 6152, 6153, 6154, 6156, 6159, 6160,
6162, 6163,
6166, 6167, 6168, 6169, 6170, 6171, 6172, 6175, 6177, 6180, 6183, 6187, 6188,
6190, 6193,
6194, 6195, 6196, 6198, 6201, 6202, 6204, 6207, 6211, 6212, 6214, 6215, 6216,
6218, 6219,
6220, 6222, 6225, 6226, 6230, 6235, 6239, 6241, 6242, 6243, 6244, 6245, 6247,
6250, 6252,
6254, 6259, 6260, 6261, 6264, 6265, 6268, 6270, 6273, 6275, 6278, 6280, 6282,
6286, 6287,
6288, 6291, 6293, 6298, 6299, 6300, 6302, 6303, 6305, 6307, 6308, 6312, 6313,
6314, 6315,
6316, 6317, 6318, 6320, 6321, 6323, 6325, 6327, 6328, 6334, 6335, 6337, 6340,
6341, 6344,
58
CA 02528853 2005-12-23
6348, 6350, 6353, 6354, 6357, 6360, 6362, 6364, 6368, 6372, 6374, 6377, 6378,
6383, 6386,
6392, 6394, 6398, 6400, 6404, 6405, 6409, 6410, 6411, 6417, 6418, 6424, 6426,
6429, 6431,
6432, 6433, 6435, 6436, 6437, 6438, 6440, 6442, 6445, 6446, 6448, 6449, 6452,
6455, 6457,
6458, 6461, 6462, 6463, 6464, 6465, 6470, 6473, 6475, 6478, 6480, 6481, 6484,
6485, 6486,
6489, 6491, 6492, 6494, 6498, 6502, 6504, 6505, 6507, 6511, 6513, 6514, 6086,
6088, 6091,
6094, 6097, 6099, 6101, 6103, 6105, 6108, 6112, 6115, 6116, 6117, 6119, 6120,
6122, 6123,
6125, 6126, 6128, 6131, 6132, 6134, 6136, 6139, 6142, 6144, 6146, 6149, 6155,
6157, 6161,
6164, 6173, 6174, 6176, 6178, 6181, 6182, 6184, 6186, 6189, 6191, 6192, 6197,
6199, 6200,
6205, 6206, 6208, 6210, 6213, 6221, 6223, 6224, 6227, 6229, 6231, 6232, 6233,
6234, 6236,
6237, 6238, 6240, 6246, 6248, 6251, 6253, 6255, 6256, 6257, 6258, 6262, 6266,
6269, 6271,
6272, 6274, 6276, 6277, 6281, 6283, 6284, 6285, 6289, 6292, 6294, 6295, 6296,
6297, 6301,
6304, 6306, 6309, 6310, 6311, 6319, 6322, 6324, 6326, 6329, 6330, 6331, 6333,
6336, 6338,
6342, 6345, 6347, 6349, 6351, 6355, 6356, 6358, 6359, 6361, 6365, 6367, 6369,
6371, 6373,
6375, 6376, 6379, 6381, 6382, 6384, 6387, 6388, 6389, 6390, 6393, 6395, 6397,
6399, 6401,
6402, 6403, 6406, 6407, 6412, 6413, 6414, 6415, 6416, 6419, 6420, 6421, 6422,
6423, 6425,
6427, 6428, 6434, 6441, 6443, 6444, 6447, 6450, 6451, 6453, 6459, 6460, 6466,
6467, 6468,
6469, 6471, 6472, 6474, 6476, 6479, 6482, 6487, 6490, 6493, 6496, 6497, 6499,
6500, 6501,
6503, 6506, 6508, 6510, 6512, 6515, 6518, 6521, 6522, 6523, 6524, 6528, 6532,
6535, 6539,
6549, 6560, 6561, 6564, 6566, 6567, 6570, 6573, 6576, 6577, 6578, 6579, 6583,
6584, 6585,
6586, 6587, 6590, 6591, 6594, 6596, 6597, 6603, 6605, 6608, 6609, 6610, 6611,
6612, 6615,
6625, 6630, 6632, 6633, 6634, 6638, 6639, 6642, 6643, 6644, 6647, 6650, 6655,
6657, 6658,
6659, 6661, 6663, 6664, 6665, 6669, 6671, 6674, 6677, 6678, 6679, 6680, 6681,
6682, 6686,
6687, 6688, 6689, 6690, 6691, 6693, 6695, 6696, 6697, 6699, 6701, 6703, 6704,
6705, 6706,
6707, 6708, 6710, 6711, 6712, 6713, 6717, 6718, 6720, 6721, 6722, 6725, 6727,
6732, 6734,
6735, 6739, 6741, 6745, 6748, 6751, 6755, 6759, 6762, 6764, 6767, 6770, 6772,
6773, 6774,
6777, 6778, 6782, 6785, 6786, 6787, 6790, 6791, 6794, 6795, 6796, 6798, 6800,
6802, 6803,
6806, 6811, 6814, 6815, 6818, 6824, 6829, 6834, 6835, 6842, 6516, 6519, 6525,
6526, 6527,
6529, 6531, 6533, 6536, 6537, 6538, 6540, 6541, 6542, 6543, 6544, 6545, 6546,
6547, 6548,
6550, 6551, 6552, 6553, 6554, 6556, 6558, 6562, 6563, 6565, 6568, 6571, 6572,
6575, 6580,
59
CA 02528853 2005-12-23
6582, 6588, 6589, 6592, 6595, 6598, 6599, 6600, 6601, 6602, 6604, 6606, 6607,
6613, 6614,
6617, 6618, 6619, 6620, 6621, 6623, 6624, 6627, 6628, 6629, 6631, 6635, 6640,
6641, 6645,
6646, 6649, 6651, 6652, 6653, 6656, 6660, 6662, 6666, 6668, 6670, 6672, 6675,
6676, 6683,
6685, 6692, 6694, 6698, 6700, 6702, 6709, 6714, 6715, 6716, 6719, 6723, 6726,
6728, 6730,
6731, 6736, 6738, 6740, 6742, 6744, 6746, 6747, 6749, 6752, 6754, 6756, 6757,
6758, 6760,
6761, 6765, 6769, 6775, 6779, 6781, 6783, 6784, 6789, 6792, 6797, 6799, 6801,
6804, 6805,
6807, 6809, 6810, 6812, 6816, 6817, 6819, 6821, 6822, 6825, 6827, 6828, 6830,
6831, 6832,
6833, 6836, 6837, 6838, 6839, 6840, 6841, 6843,
Detection: SEQ ID NOS:6517, 6520, 6530, 6534, 6555, 6557, 6559, 6569, 6574,
6581,
6593, 6616, 6622, 6626, 6636, 6637, 6648, 6654, 6667, 6673, 6684, 6724, 6729,
6733, 6737,
6743, 6750, 6753, 6763, 6766, 6768, 6771, 6776, 6780, 6788, 6793, 6808, 6813,
6820, 6823,
6826, 6844y 6848, 6850, 6852, 6854, 6857, 6859, 6861, 6862, 6865, 6868, 6870,
6871, 6872,
6874, 6875, 6876, 6880, 6883, 6886, 6887, 6888, 6890, 6891, 6894, 6896, 6897,
6898, 6899,
6900, 6901, 6902, 6904, 6905, 6906, 6910, 6912, 6917, 6920, 6845, 6847, 6849,
6851, 6853,
6855, 6858, 6860, 6863, 6866, 6869, 6873, 6877, 6879, 6881, 6882, 6884, 6889,
6892, 6893,
6895, 6907, 6908, 6909, 6911, 6913, 6915, 6916, 6918, 6921, 6924, 6927, 6928,
6933, 6935,
6937, 6939, 6940, 6943, 6946, 6947, 6948, 6949, 6951, 6953, 6954, 6955, 6959,
6961, 6964, .
6965, 6968, 6969, 6972, 6973, 6976, 6979, 6980, 6922, 6925, 6929, 6930, 6931,
6932, 6934,
6936, 6942, 6944, 6945, 6950, 6952, 6956, 6957, 6958, 6960, 6962, 6963, 6966,
6967, 6970,
6971, 6974, 6975, 6977, 6981, 6983, 6986, 6992, 6995, 6998, 7002, 7006, 7013,
7017, 7022,
7024, 7027, 7030, 7032, 7036, 7037, 7040, 7050, 7052, 7055, 7056, 7058, 7059,
7060, 7064,
7067, 7072, 7074, 7076, 7077, 7084, 7085, 7086, 7087, 7088, 7089, 7090, 7091,
7096, 7097,
7100, 7102, 7109, 7110, 7113, 7114, 7115, 7118, 7121, 7123, 7124, 7131, 7133,
7135, 7136,
7138, 7139, 7140, 7144, 7146, 7148, 7149, 7158, 7159, 7160, 7163, 7165, 7166,
7168, 7172,
7175, 7178, 7180, 7181, 7183, 7185, 7186, 7187, 7192, 7194, 7199, 7202, 7203,
7204, 7205,
7207, 7208, 7213, 7214, 7216, 7220, 7221, 7227, 7228, 7230, 7233, 7235, 7237,
7239, 7243,
7245, 7246, 7248, 7249, 7251, 7255, 7256, 7260, 7263, 7264, 7265, 7266, 7268,
7269, 7272,
7276, 7277, 7278, 7279, 7280, 7281, 7283, 7284, 7287, 7291, 7295, 7299, 7302,
7303, 7306,
7309, 7310, 7311, 7314, 7315, 7320, 7321, 7324, 7325, 7329, 7331, 7332, 7334,
7336, 7339,
19
'~ILL 'ZILL 'I ILL '60LL 'LOLL 'VOLL 'ZOLL 'IOLL '669L 'K)9L '169L '0691 '689L
'L89L 'S89L
'~89L 'Z89L 'LL9L '9L9L 'tL9L 'ZL9L '999L '199L 'S99L 'I S9L 'Lti9L '~b9L
'9C9L S~9L '~~9L
'6Z9L 'LZ9L '~Z9L 'IZ9L 'OZ9L '819L '9i9L '0I9L '609L '909L 'CO9L '009L '86SL
'L65L 496SL
'06SL '68SL '885L 'L85L '98SL 'SBSL 'ZBSL 'I8SL '08SL '6L9L '8L5L 'LLSL '9LSL
'SLSL 'nSL
'~LSL ILSL 'OLSL 'L95L '999L '599L '095L '69SL '895L 'SSSL 'OSSL 'BbSL 'Lt~SL
'9bSL 'bbSL SZ
'i'6SL '6~SL '8~SL 'L~SL '9~SL 'S~SL 'KSL '~~SL '0~SL '9ZSL '~ZSL 'IZ5L 'LISL
'9I5L 'SISL
'~ISL 'I ISL 'OISL '605L 'LOSL '909L '90SL 'IOSL '66tiL '86bL 'L6bL 'S6tIL
'b6tiL 'Z6'bL '88bL
UtiL 't,8VL '~BVL 'Z8tL '6LbL '8LtiL 'SLtiL 'ZL'bL 'ML 119ft '094L 'LStL 'SS~L
'PStt '~SVL
'ZSbL 'I01L 'OStbL 'LttL '9trbL 'S"L '9~t'L 't~bL '~~tL 'Z~tiL 'IEt~L '0~7L
'8ZbL '9ZbL '~ZbL
'tiS5L 'ZSSL 4 6VSL 'StlSL '~#?SL 'ZtrSL 'ObSL 'Z~SL '6ZSL '8ZSL 'LZSL 'SZSL
'VZSL 'ZZSL 'OZSL 0Z
'6I9L '8I54 'ZISL '809L '~0SL '00SL '96t~L 'E6tL '06tL '68tiL '98bL '58bL
'I8bL '08tL 'LLbL
'9LbL $L1lL 'ILbL 'OLtiL 'L9pL '99tL '99tiL '~9bL 'Z9tL '69ft '9SbL '6ttL
'81bbL 'b"L 'Zt'bL
'0'aVL '6~tiL '801L 'S~VL 'LZtlL 'SZtL 'ZZbL 'IZtiL '6ItiL 'IItiL '60tiL
'80'PL 'LObL '1oL '~OtrL
'00bL '86~L '96~L '~6~Z '68EL '88~L '98EL 'fi8~L '~8~L 'Z8~L '6L~L '9L~L '~L~L
'IL~L 'OL~L
'69~L '89~L 'L9~L '99~L 'S9~L 'V9~L '~9~L '19~L '69~L 'LS~L '9S~L 'fi5~L 'IS~L
'01EL '8b~L SI
'L'b~L 'ML 'Ztr~L 'L~~L 'S~~L 'EEEL '0~~L '8Z~L '9Z~L 'ZZ~L '61~L '$i~L 'LI~L
'9I~L 'ZIEL
'80~L 'LO~L '1~0~L 'IO~L '86ZL 'L6ZL '96ZL 'i+6ZL '~6ZL 'Z6ZL '06ZL '68ZL
'SBZL 'ZBZL 'SLZL
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CA 02528853 2005-12-23
7714, 7716, 7718, 7720, 7726, 7728, 7729, 7733, 7734, 7735, 7736, 7738, 7740,
7741, 7744,
7746, 7747, 7748, 7749, 7750, 7751, 7752, 7753, 7754, 7755, 7756, 7758, 7556,
7561, 7563,
7568, 7583, 7584, 7591, 7593, 7594, 7595, 7599, 7601, 7604, 7607, 7608, 7611,
7613, 7614,
7616, 7617, 7619, 7622, 7624, 7626, 7628, 7631, 7637, 7638, 7639, 7640, 7641,
7642, 7644,
7645, 7646, 7649, 7650, 7652, 7654, 7656, 7657, 7658, 7659, 7660, 7662, 7663,
7665, 7667,
7669, 7670, 7671, 7673, 7675, 7678, 7679, 7680, 7681, 7684, 7686, 7688, 7692,
7695, 7697,
7698, 7700, 7703, 7705, 7708, 7710, 7715, 7717, 7719, 7721, 7722, 7723, 7724,
7725, 7727,
7730, 7731, 7732, 7737, 7739, 7742, 7743, 7745, 7757, 7759, 7760, 7761, 7764,
7770, 7774,
7775, 7778, 7782, 7786, 7788, 7795, 7797, 7799, 7805, 7807, 7809, 7810, 7811,
7815, 7817,
7819, 7820, 7822, 7823, 7824, 7828, 7832, 7834, 7837, 7839, 7843, 7845, 7851,
7852, 7857,
7860, 7863, 7865, 7868, 7874, 7881, 7885, 7889, 7892, 7894, 7896, 7900, 7902,
7904, 7907,
7909, 7910, 7913, 7917, 7922, 7925, 7926, 7928, 7933, 7934, 7937, 7941, 7945,
7946, 7948,
7950, 7951, 7952, 7953, 7954, 7955, 7956, 7957, 7963, 7964, 7968, 7971, 7972,
7973, 7976,
7979, 7980, 7981; 7982, 7983, 7985, 7986, 7987, 7988, 7991, 7762, 7765, 7767,
7768, 7771,
7773, 7776, 7779, 7781, 7783, 7784, 7785, 7789, 7791, 7792, 7793, 7794, 7796,
7798, 7800,
7802, 7803, 7808, 7812, 7813, 7814, 7816, 7818, 7821, 7825, 7827, 7829, 7830,
7833, 7835;
7836, 7838, 7840, 7841, 7842, 7844, 7846, 7847, 7848, 7849, 7853, 7854, 7855,
7856, 7858,
7861, 7864, 7866, 7869, 7870, 7871, 7872, 7873, 7875, 7877, 7878, 7879, 7880,'
7882, 7884,
7886, 7888, 7890, 7897, 7899, 7903, 7905, 7912, 7914, 7916, 7918, 7919, 7920,
7921, 7923,
7929, 7931, 7932, 7936, 7938, 7939, 7940, 7942, 7943, 7944, 7947, 7949, 7958,
7960, 7962,
7965, 7967, 7969, 7970, 7974, 7977, 7989, 7990, 7992; 7995, 7996, 8000, 8003,
8006, 8007;
8010, 8012, 8015, 8016, 8018, 8019, 8026, 8027, 8030, 8034, 8035, 8038, 8039,
8043, 8044,
8046, 8049, 8050, 8052, 8053, 8055, 8056, 8062, 8064, 8066, 8067, 8068, 8069,
8070, 8071,
8074, 8076, 8077, 8078, 8079, 8080, 8081, 7993, 7997, 7999, 8001, 8004, 8005,
8008, 8009,
8011, 8013, 8017, 8020, 8021, 8022, 8023, 8024, 8025, 8028, 8031, 8033, 8036,
8040, 8042,
8047, 8051, 8054, 8057, 8059, 8060, 8061, 8065, 8072, 8073, 8075, 8082, 8083,
8084, 8085,
8087, 8088, 8089, 8090, 8093, 8095, 8098, 8100, 8101, 8102, 8104, 8107, 8110,
8111, 8114,
8115, 8116, 8118, 8122, 8124, 8126, 8127, 8129, 8132, 8134, 8136, 8138, 8142,
8143, 8144,
8146, 8151, 8157, 8160, 8163, 8165, 8168, 8169, 8170, 8173, 8174, 8175, 8176,
8178, 8181,
62
CA 02528853 2005-12-23
8185, 8187, 8188, 8189, 8190, 8191, 8193, 8194, 8200, 8203, 8206, 8208, 8209,
8212, 8214,
8216, 8217, 8220, 8222, 8224, 8228, 8230, 8091, 8094, 8096, 8099, 8103, 8105,
8108, 8112,
8113, 8117, 8119, 8121, 8123, 8125, 8128, 8130, 8131, 8133, 8135, 8137, 8139,
8145, 8147,
8148, 8149, 8150, 8152, 8153, 8154, 8155, 8156, 8158, 8161, 8164, 8166, 8167,
8171, 8177,
8179, 8180, 8182, 8183, 8184, 8186, 8192, 8195, 8197, 8198, 8199, 8201, 8202,
8204, 8207,
8210, 8213, 8218, 8221, 8223, 8225, 8227, 8229, 8231, 8232, 8235, 8236, 8237,
8238, 8244,
8245, 8247, 8250, 8251, 8253, 8254, 8255, 8256, 8258, 8259, 8260, 8263, 8267,
8269, 8270,
8273, 8274, 8277, 8280, 8281, 8289, 8292, 8293, 8294, 8295, 8296, 8297, 8298,
8301, 8305,
8306, 8310, 8311, 8312, 8315, 8317, 8318, 8322, 8323, 8324, 8327, 8328, 8329,
8330, 8333,
8336, 8339, 8340, 8347, 8349, 8350, 8352, 8353, 8356, 8359, 8360, 8366, 8369,
8370, 8372,
8374, 8376, 8377, 8379, 8381, 8382, 8383, 8233, 8239, 8240, 8242, 8243, 8246,
8248, 8252,
8257, 8261, 8264, 8266, 8268, 8271, 8275, 8278, 8282, 8284, 8285, 8286, 8287,
8288, 8291,
8299, 8302, 8303, 8304, 8307, 8309, 8313, 8314, 8316, 8319, 8321, 8325, 8326,
8331, 8332,
8334, 8335, 8337, 8341, 8342, 8343, 8344, 8345, 8346, 8348, 8351, 8354, 8357,
8361, 8362,
8363, 8364, 8365, 8367, 8371, 8373, 8375, 8378, 8384, 8387, 8390, 8391, 8397,
8398, 8401,
8402, 8405, 8406, 8409, 8414, 8418, 8419, 8422, 8425, 8428, 8431, 8432, 8433,
8435, 8437,.
8440, 8444, 8449, 8452, 8454, 8455, 8457, 8462, 8464, 8466, 8468, 8469, 8472,
8475, 8476, =
8478, 8479, 8481, 8484, 8487, 8488, 8490, 8493, 8496, 8497, 8498, 8501, 8505,
8508, 8511,
8513, 8515, 8517, 8518, 8519, 8521, 8522, 8523, 8526, 8528, 8530, 8531, 8532,
8534, 8536,
8541, 8544, 8547, 8551, 8554, 8556, 8558, 8560, 8562, 8563, 8566, 8567, 8568,
8570, 8571,
8574, 8575, 8576, 8577, 8580, 8581, 8585, 8588, 8591, 8594, 8601, 8602, 8603,
8608, 8609,
8613, 8614, 8617, 8618, 8619, 8620, 8623, 8624, 8628, 8629, 8630, 8633, 8634,
8635, 8636,
8638, 8643, 8645, 8648, 8654, 8657, 8658, 8659, 8660, 8661, 8662, 8666, 8670,
8672, 8674,
8676, 8681, 8683, 8686, 8688, 8689, 8691, 8692, 8693, 8695, 8696, 8700, 8701,
8702, 8703,
8704, 8705, 8709, 8716, 8717, 8719, 8722, 8725, 8727, 8728, 8729, 8732, 8733,
8734, 8735,
8736, 8385, 8388, 8392; 8394, 8395, 8399, 8403, 8407, 8410, 8411, 8412, 8413,
8415, 8416,
8420, 8423, 8424, 8426, 8429, 8434, 8436, 8438, 8442, 8445, 8446, 8447, 8448,
8450, 8451,
8456, 8458, 8460, 8461, 8463, 8465, 8467, 8470, 8473, 8474, 8477, 8480, 8482,
8485, 8486,
8489, 8491, 8492, 8494, 8499, 8500, 8502, 8503, 8504, 8506, 8510, 8512, 8514,
8516, 8520,
63
CA 02528853 2005-12-23
8524, 8527, 8529, 8533, 8S35, 8537, 8539, 8542, 8545, 8546, 8548, 8550, 8552,
8555, 8557,
8564, 8565, 8569, 8572, 8578, 8582, 8583, 8586, 8587, 8589, 8592, 8595, 8596,
8597, 8598,
8599, 8600, 8604, 8606, 8607, 8610, 8612, 8615, 8616, 8621, 8625, 8627, 8631,
8632, 8637,
8639, 8640, 8641, 8642, 8644, 8646, 8647, 8649, 8650, 8651, 8652, 8653, 8655,
8663, 8665,
8667, 8668, 8669, 8671, 8673, 8675, 8677; 8678, 8679, 8680, 8682, 8684, 8685,
8687, 8690,
8694, 8697, 8698, 8699, 8706, 8707, 8708, 8710, 8711, 8712, 8713, 8714, 8715,
8718, 8720,
8723, 8726, 8730, 8737, 8738, 8741, 8744, 8747, 8750, 8751, 8753, 8754, 8755,
8756, 8759,
8760, 8762, 8763, 8766, 8767, 8768, 8769, 8770, 8772, 8775, 8776, 8777, 8779,
8782, 8783,
8784, 8785, 8786, 8787, 8788, 8789, 8790, 8793, 8794, 8796, 8798, 8799, 8801,
8803, 8804,
8806, 8807, 8809, 8812, 8813, 8815, 8820, 8823, 8826, 8829, 8830, 8832, 8835,
8836, 8837,
8842, 8845, 8846, 8847, 8851, 8853, 8856, 8857, 8859, 8861, 8866, 8739, 8878,
8879, 8880,
8884, 8891, 8893, 8895, 8898, 8900, 8901, 8909, 8912, 8739, 8742, 8745, 8748,
8749, 8752,
8757, 8758, 8761, 8764, 8771, 8773, 8778, 8780, 8791, 8797, 8800, 8802, 8805,
8808, 8810;
8814, 8816, 8818, 8819, 8821, 8824, 8827, 8828, 8831, 8833, 8838, 8840, 8841,
8755, 8843,
8844, 8848, 8850, 8852, 8854, 8855, 8863, 8864, 8865, 8867, 8868, 8869, 8870,
8871, 8872,
8873, 8874, 8876, 8877, 8881, 8882, 8883, 8885, 8887, 8888, 8889, 8890, 8892,
8894, 8896,
8897, 8899, 8902, 8785, 8903, 8904, 8905, 8906, 8907, 8908, 8911, 8913, 8917,
8920, 8923,
8924, 8929, 8931, 8934, 8935, 8936, 8914, 8916, 8918, 8921, 8925, 8927, 8928,
8930, 8932,
8933, 8937, 8980, 8984, 8988, 8989, 8998, 9001, 9002, 9005, 9007, 9008, 9012,
9013, 9016,
9019, 9020, 9022, 9023, 9025, 9028, 9029, 9032, 9033, 9034, 9035, 9037, 9038,
9044, 9046,
9047, 9048, 9049, 9054, 8981, 8983, 8985, 8987, 8990, 8992, 8993, 8994, 8995,
8997, 8999,
9003, 9004, 9006, 9009, 9010, 9011, 9014, 9017, 9018, 9021, 9024, 9026, 9030,
9036, 9039,
9041, 9042, 9043, 9045, 9050, 9051, 9052, 9053, 9055, 9058, 9060, 9063, 9066,
9067, 9068,
9069, 9070, 9071, 9074, 9075, 9077, 9078, 9079, 9080, 9082, 9084, 9088, 9089,
9090, 9099,
9103, 9104, 9105, 9107, 9111, 9056, 9061, 9064, 9072, 9081, 9083, 9085, 9086,
9087, 9091,
9092, 9093, 9094, 9095, 9096, 9097, 9098, 9100, 9102, 9106, 9108, 9109, 9110,
9112, 9113,
9116, 9118, 9125, 9126, 9128, 9129, 9130, 9136, 9141, 9143, 9148, 9150, 9153,
9157, 9158,
9159, 9163, 9164, 9165, 9167, 9168, 9171, 9173, 9177, 9180, 9181, 9182, 9183,
9185, 9187,
9188, 9189, 9191, 9192, 9193, 9194, 9197, 9198, 9200, 9206, 9207, 9208, 9209,
9211, 9216,
64
CA 02528853 2005-12-23
9218, 9219, 9220, 9221, 9222, 9223, 9226, 9228, 9229, 9230, 9231, 9232, 9233,
9234, 9237,
9238, 9240, 9243, 9246, 9248, 9253, 9256, 9257, 9260, 9263, 9264, 9266, 9269,
9271, 9279,
9280, 9281, 9282, 9283, 9284, 9290, 9114, 9119, 9121, 9122, 9123, 9124, 9127,
9131, 9133,
9134, 9135, 9137, 9138, 9139, 9140, 9142, 9144, 9146, 9147, 9149, 9151, 9152,
9154, 9155,
9156, 9160, 9162; 9166, 9169, 9172, 9174, 9175, 9176, 9178, 9184, 9186, 9190,
9195, 9199,
9201, 9202, 9203, 9204, 9205, 9212, 9214, 9215, 9217, 9224, 9227, 9235, 9239,
9241, 9244,
9247, 9249, 9251, 9252, 9254, 9258, 9261, 9265, 9267, 9270, 9273. 9274, 9275,
9276, 9277,
9278, 9285, 9286, 9287, 9288, 9289, 9291, 9292, 9295, 9297, 9300, 9302, 9303,
9304, 9307,
9308, 9310, 9311; 9313, 9319, 9321, 9323, 9324, 9328, 9333, 9334, 9337, 9338,
9339, 9342,
9344, 9348, 9351, 9352, 9354, 9355, 9357, 9360, 9367, 9371, 9374, 9377, 9379,
9383, 9384,
'9385, 9388, 9389, 9390, 9393, 9394, 9398, 9401, 9402, 9406, 9408, 9409, 9410,
9413, 9415,
9416, 9417, 9418, 9419, 9421, 9422, 9425, 9429, 9433, 9434, 9435, 9436, 9437,
9439, 9440,
9446, 9452, 9453, 9454, 9455, 9457, 9458, 9459, 9462, 9466, 9467, 9468, 9470,
9474, 9479,
.9482, 9487, 9488, 9489, 9492, 9495, 9497, 9499, 9500, 9504, 9505, 9508, 9513,
9514, 9519,
9522, 9523, 9524, 9526, 9528, 9532, 9533, 9534, 9535, 9537, 9538, 9541, 9542,
9543, 9545,
9546, 9548, 9293, 9296, 9298, 9301, 9305, 9309, 9312, 9314, 9316, 9317, 9318,
9320, 9322,
9325, 9326, 9327, 9329, 9331, 9332, 9335, 9336, 9340, 9345, 9347, 9349, 9353,
9356, 9358,
9361, 9363, 9364, 9365, 9368, 9370, 9372, 9375, 9376, 9378, 9380, 9382, 9386,
9391, 9395,
9397, 9399, 9400, 9403, 9405, 9407, 9411, 9412, 9414, 9420, 9423, 9424, 9426,
9428, 9430,
9431, 9432, 9438, 9441, 9443, 9444, 9445, 9447, 9448, 9449, 9450, 9451, 9456,
9460, 9461,
9464, 9465, 9469, 9471, 9473, 9475, 9477, 9478, 9480, 9483, 9485, 9490, 9493,
9494, 9496,
9498, 9501, 9502, 9503, 9506, 9509, 9511, 9512, 9515, 9516, 9517, 9518, 9520,
9521, 9525,
9527, 9529, 9530, 9536, 9539, 9544, 9547, 9549, 9550, 9551, 9555, 9556, 9557,
9559, 9560,
9563, 9564, 9565, 9566, 9569, 9570, 9573, 9577, 9578, 9579, 9580, 9585, 9586,
9587, 9588,
9591, 9593, 9596, 9552, 9554, 9558, 9561, 9567, 9571, 9574, 9576, 9581, 9583,
9584, 9589,
9592, 9594, 9595, 9598, 9602, 9604, 9605, 9608, 9612, 9613, 9614, 9617, 9618,
9621, 9622,
9625, 9626, 9627, 9632, 9633, 9638, 9640, 9643, 9644, 9645, 9646, 9651, 9654,
9656, 9657,
9658, 9661, 9662, 9665, 9666, 9667, 9668, 9672, 9673, 9675, 9676, 9677, 9680,
9683, 9686,
9688, 9691, 9694, 9698, 9699, 9702, 9705, 9707, 9710, 9711, 9715, 9719, 9720,
9722, 9723,
CA 02528853 2005-12-23
9726, 9729, 9732, 9733, 9735, 9737, 9738, 9741, 9744, 9745, 9746, 9747, 9748,
9749, 9750,
9754, 9755, 9756, 9758, 9759, 9768, 9771, 9772, 9776, 9777, 9781, 9782, 9784,
9787, 9789,
9790, 9792, 9796, 9797, 9800, 9805, 9806, 9812, 9815, 9818, 9821, 9823, 9825,
9827, 9831,
9832, 9835, 9837, 9839, 9841, 9842, 9844, 9845, 9851, 9853, 9854, 9855, 9857,
9860, 9862,
9863, 9865, 9871, 9873, 9875, 9878, 9879, 9599, 9601, 9603, 9606, 9607, 9609,
9611, 9615,
9619, 9623, 9624, 9628, 9630, 9631, 9634, 9635, 9637, 9639, 9641, 9647, 9649,
9650, 9652,
9653, 9659, 9660, 9663, 9664, 9670, 9671, 9679, 9682, 9684, 9687, 9690, 9692,
9693, 9695,
9697, 9700, 9701, 9703, 9706, 9708, 9712, 9713, 9714, 9716, 9717, 9718, 9721,
9724, 9727,
9730, 9734, 9739, 9742, 9751, 9753, 9757, 9760, 9761, 9762, 9763, 9764, 9765,
9766, 9767, .
9769, 9773, 9775, 9778, 9780, 9783, 9786, 9788, 9791, 9793, 9794, 9795, 9798,
9801, 9803,
9804, 9807, 9808, 9809, 9810, 9811, 9813, 9816, 9817, 9819, 9820, 9822, 9826,
9828, 9830,
9833, 9836, 9838, 9840, 9843, 9846, 9847, 9848, 9849, 9850, 9852, 9856, 9858,
9859, 9861,
9864, 9866, 9867, 9868, 9869, 9870, 9872, 9876, 9877, 9880, 9881, 9882, 9886,
9890, 9891,
9894, 9900, 9902, 9904, 9908, 9911, 9912, 9915, 9917, 9919, 9923, 9925, 9928,
9935, 9936,
9940, 9943, 9949, 9953, 9954, 9956, 9957, 9958, 9962, 9963, 9964, 9968, 9969,
9975, 9978,
9981, 9988, 9989, 9990, 9991, 9992, 9995, 9997, 10011, 10012, 10014, 10017,
10018, 10031,
10032, 10033, 10034, 10035, 10037, 10041, 10042, 10046, 10050, 10054, 10058,
10059, 9883,
9885, 9887, 9889, 9892, 9893, 9895, 9896, 9897, 9898, 9899, 9901, 9903, 9905,
9907, 9909,
9913, 9914, 9916, 9918, 9920, 9921, 9922, 9924, 9926, 9929, 9931, 9932, 9933,
9934, 9937,
9938, 9939, 9941, 9942, 9944, 9945, 9946, 9947, 9948, 9950, 9952, 9955, 9959,
9960, 9961,
9966, 9967, 9970, 9972, 9973, 9974, 9976, 9979, 9980, 9982, 9983, 9984, 9985,
9986, 9987,
9993, 9994, 9996; 9998, 9999, 10000, 10001, 10002, 10003, 10004, 10005, 10006,
10007,
10008, 10009, 10010, 10013,10015, 10016, 10019, 10020, 10021, 10022, 10023,
10025, 10026,
10027, 10028, 10030, 1003 6, 1003 8, 10040, 10043, 10044, 10045, 10047, 10048,
10049, 10051,
10053,10055, 10056, 10057, 10060, 10061, 10064, 10067, 10068, 10070, 10071,
10072, 10074,
10079, 10081, 10083, 10088, 10091, 10095, 10096, 10097, 10101, 10103, 10105,
10106, 10109,
10110, 10111, 10113, 10116,10118,10120,10122,10125, 10126,10127,10131, 10134,
10137,
10138, 10144, 10147, 10149,10151,10152, 10155, 10156, 10i59, 10160,10169,
10172, 10174,
10175, 10178, 10181, 10186,10188,10189,10190, 10191, 10192, 10196,
10199,10203, 10204,
66
CA 02528853 2005-12-23
10210,10211,10216, 10218, 10221, 10224, 10225, 10226, 10229, 10230, 10231,
10232, 10234,
10239, 10241,10244, 10252, 10253,10257, 10258, 10259, 10261, 10266,10272,
10276, 10062,
10065, 10069, 10075, 10077,10078,10080,10082, 10084, 10086, 10087,10089,
10090, 10092,
10093, 10094, 10098, 10099, 10100, 10102, 10104, 10107, 10112, 10114, 10117,
10119, 10121,
10123, 10124, 10129, 10130,10132,10133, 10135, 10139, 10140,10141, 10142,
10143, 10145,
10148, 10150, 10153, 10154, 10157, 10161, 10163, 10164, 10165,10166,
10167,10170, 10173,
10176, 10177, 10179, 10182, 10183, 10184, 10187, 10193, 10194, 10195, 10197,
10198,10200,
10201, 10202, 10205, 10206, 10207, 10209, 10212, 10213, 10214, 10215, 10219,
10220, 10222,
10223, 10227, 10228, 10233, 10235, 10236, 10237, 10240, 10242, 10243, 10245,
10247, 10248,
10249, 10251, 10254, 10255, 10256,10260,10262, 10263, 10264, 10265, 10267,
10268, 10269,
10270, 10271, 10273, 10274, 10275, 10277, 10278, 10279, 10282, 10285, 10287,
10291, 10292,
10297, 10299, 10301, 10304, 10306, 10308, 10310, 10314, 10317, 10318, 10320,
10322, 10327,
10329, 10330, 10333, 10335, 10336, 10337, 10340, 10342, 10348, 10350, 10351,
10354, 10358,
10359, 10360, 10362, 10366, 10369, 10372, 10373, 10374, 10376, 10378, 10383,
10385, 10391,
10398, 10403, 10407, 10410, 10413, 10415, 10419, 10422, 10425, 10427, 10428,
10429, 10430,
10432, 10433, 10434, 10437, 10438, 10439, 10442, 10443, 10445, 10446, 10447,
10448, 10452,
10453, 10454, 10455, 10456, 10460,10463,10466, 10467, 10469, 10470, 10473,
10475, 10477,
10480, 10484, 10488, 10490, 10494, 10495, 10497, 10501, 10502, 10503, 10507,
10508, 10510,
10517, 10518, 10520, 10522, 10524,10525, 10528, 10529, 10533, 10536, 10537,
10540, 10541,
10545, 10547, 10551, 10553, 10556, 10559, 10560, 10563, 10564, 10565, 10568,
10571, 10574,
10576,10579, 10581, 10583,10588,10589, 10590, 10592,10593, 10596, 10597,10601,
10602,
10603, 10605, 10607, 10609, 10610,10612, 10613, 10617, 10618, 10621,10614,
10627, 10630,
10632,10634, 10637, 10638, 10639,10640,10641, 10643, 10645, 10647, 10653,
10655, 10657,
10659, 10661, 10662, 10663, 10664, 10665, 10666, 10667, 10668, 10669, 10672,
10673, 10674,
10675, 10280, 10283, 10286, 10288, 10290, 10293, 10295, 10298, 10300, 10302,
10305, 10307,
10309,10311, 10312, 10313, 10315, 10319, 10321, 10323,10325,10326,10328,
10331, 10338,
10341, 10343, 10344, 10345, 10346, 10347, 10349, 10352, 10355, 10356, 10357,
10361, 10363,
10365, 10367, 10370, 10375, 10377, 10379, 10380, 10381, 10382, 10384, 10386,
10387, 10388,
10390, 10392, 10394, 10395, 10399,10401, 10402, 10404, 10406, 10408, 10411,
10412, 10414,
67
CA 02528853 2005-12-23
10416, 10417,10418,10420,10423, 10426,10431, 10435, 10440,10444, 10449, 10451,
10457,
10459,10461, 10462, 10464, 10465, 10468, 10471, 10476, 10478, 10481, 10483,
10485, 10487,
10489,10491,10492,10493,10498,10499,10500,10504, 10506,10509,
10511,10513,10514,
10515,10516,10519,10521,
10523,10526,10530,10531,10532,10534,10535,10538,10539,
10542,10543,10544,10546,10548,10550,10552,10554,10557,10558,10561,10566,10569,
10572,10573,10577, 10578,10580,10582,
10584,10586,10594,10598,10600,10604,10606,
10611,10614,10616,10619,10622,10625,
10626,10628,10631,10633,10635,10642,10644,
10646,10648,10649,10650, 10651,10652,10654, 10656,
10658,10660,10670,10671,10676,
10678, 10684,10679,10681, 10682,10683, 10685,10687,10688, 10689,
10690,10691,10693,
10696,10699,10703, 10704, 10709, 10711, 10712, 10713, 10720,10721,
10722,10724,10725,
10726,10728,10729, 10734,10740, 10742, 10743,10744,10745,10746,
10748,10749,10751,
10754,10758, 10759, 10760, 10764, 10768, 10769,10772, 10775, 10780, 10781,
10783,10786,
10791,10796,10801,10808, 10817,10818, 10819,10821, 10823, 10826,
10827,10829,10830,
10834,10843, 10844, 10847, 10848, 10849, 10853,10856, 10858,10866, 10869,
10872,10874,
10877,10878,10879,10882, 10884,10885, 10889,10892, 10893,10896,10900,
10901,10903,
10905,10907, 10909, 10910, 10911,10914, 10916,10919, 10926,10930, 10932,
10933,10934,
10938, 10941, 10942, 10943, 10944,10946, 10947,10953, 10955, 10956, 10958,
10959, 10962,
10967,10968, 10972, 10975, 10977,10978,10981,10984, 10985,10988, 10992,10995,
10997,
10999, 11000, 11001, 11006, 11009,11013, 11016,11020,11025, 11033, 11036,
11040,11041,
11042, 11043, 11045, 11046, 11048, 11052, 11055,11060, 11061,11062, 11064,
11065,11067,
11070, 11071, 11072, 11078, 11079, 11081,11085,11093, 10694, 10697, 10700,
10702, 10705,
10707,10710, 10714, 10716, 10717,10718,10719,10723, 10727,10730, 10732,10733,
10735,
10737,10738, 10739, 10741,10747,10750, 10752, 10755, 10757,10761, 10762,
10763, 10765,
10767, 10770, 10773, 10776, 10778,10779, 10782, 10784,10788, 10789,
10790,10792, 10793,
10794, 10798, 10799, 10800, 10802, 10804, 10805, 10806, 10807, 10809, 10811,
10812, 10813,
10814, 10815, 10816, 10820, 10822, 10824, 10831, 10833, 10835, 10837, 10838,
10839, 10840,
10841,10842,10845, 10846, 10850,10851,10854, 10857, 10859,10860, 10861,10862,
10863,
10864, 10865, 10868, 10870, 10871, 10873, 10875, 10880, 10881, 10883, 10886,
108 88, 10890,
10894, 10897, 10899, 10902, 10904, 10906, 10908, 10912, 10915, 10917, 10920,
10922, 10924,
68
CA 02528853 2005-12-23
10925, 10927, 10928, 10929,10931, 10935, 10937,10939,10945, 10948,
10950,10951, 10952,
10954,10957,10960,10963, 10965, 10966, 10969,10970,10971, 10973, 10976,
10979,10980,
10982, 10986,10987,10989,10991,10993,10996,10998,11002,11004, 11005, 11007,
11010,
11012,11014,11015,11017,11019,11021,11022,11023,11026,11027,
11028,11029,11030,
11031, 11032, 11034,11037, 11039,11044,11047, 11049, 11050,11051,11053,
11054,11056,
11058, 11059,11063, 11066, 11068,11069, 11073,11074,11075,
11076,11077,11080,11082,
11083, 11084,11086,11087, 11088,11089,11090,11091,11092,11094,
11376,11381,11382,
11383, 11384, 11387,11397, 11401,11405,11408, 11414,11418, 11421, 11422,
11424,11427,
11429,11430,11377,11379,11380,11385,11386,11388,11390,11391,
11392,11394,11395,
11396,11398,11399,11400, 11402,11403, 11404,11406,11409, 11410, 11411,11412,
11415,
11416, 11417,11419,11423,11425,11428, 11431, 11432,11433,
11434,11438,11442,11443,
11447,11449,11450, 11452,11456,11459, 11463,11464,11467, 11468, 11472,11477,
11480,
11481,11482, 11483,11484, 11488, 11489,11493,11498, 11502, 11504, 11505,
11506,11507,
11508,11509, 11512, 11513, 11514, 11517,11520, 11522,11523, 11524,
11526,11528,11532,
11534,11435, 11439, 11444,11445, 11446, 11448,11451, 11453, 11457, 11458,
11460, 11461,
11462, 11465,11466, 11469, 11473, 11474,11475,11476, 11478, 11479,
11485,11486,11487;
11490,11494,11497, 11499, 11500,11501,11503,11510, 11511,11515, 11518,11519,
11521,
11525, 11527,11529, 11535,11539,11540,11541,11548,11550, 11551, 11552, 11555,
11556,
11557,11560, 11561, 11562, 11568, 11569, 11571,11572, 11573, 11577, 11536,
11542, 11545,
11546, 11547, 11549, 11553, 11554, 11563, 11566,11570,11574, 11578, 11585,
11586, 11590,
11591,11592,11595, 11596, 11597,11600, 11579,11582, 11587,11593, 11594,
11598,11601,
11602, 11606, 11607, 11608,11609,11616,11618,11622, 11626, 11628, 11632,
11633, 11603,
11610, 11613,11614, 11615,11617,11619,11623,11627, 11629,11634, 11635, 11640,
11641,
11642, 11643, 11647, 11648, 11654, 11655, 11656, 11657, 11658,11636,11639,
11644,11645,
11646, 11649, 11652, 11653,14659, 11663, 11664,11665, 11666, 11670,
11671,11672, 11673,
11677, 11681, 11687, 11690, 11694, 11696, 11697,11701, 11705,11709, 11713,
11719, 11723,
11729, 11733, 11739, 11744, 11745, 11748, 11749, 11751, 11755,11759, 11763,
11770, 11772,
11774,11776,11778,11782,11785,11660,11667,11674,11675,11676,
11678,11682,11685,
11686, 11688,11689,11691,11692,11693,11695,11698,11699,11'100,11702,
11706,11710,
69
CA 02528853 2005-12-23
11716,11720,11724, 11725, 11727,11728,11730,11731,11732, 11734, 11735,
11736,11737,
11738,11740,11743, 11746,11747,11750,11752, 11756,11761, 11762, 11764,11765,
11767,
11768,11769, 11773, 11775, 11777, 11779, 11783, 11784, 11786, 11790, 11791,
11792, 11787,
11793, 11796, 11800, 11802, 11803, 11805, 11810, 11818, 11819, 11823, 11824,
11827, 11828,
11829,11833,11836,11837,11839,11840,11847,11850,11852,11857,11860,11864,11866,
11797,11801, 11804, 11806, 11809, 11811, 11814,11815, 11817, 11820, 11825,
11830, 11835,
11841, 11844, 11845, 11846, 11848, 11851, 11853, 11856, 11858, 11861, 11862,
11863, 11865,
11867,11871,11876,
11878,11882,11888,11891,11894,11868,11872,11875,11877,11879,
11883,11886, "11887, 11889,11890,11892,11893, 11895, 11898, 11902, 11903,
11904, 11908,
11909,11911,11912, 11913, 11914,11915, 11917, 11918, 11922,11926, 11927,
11930, 11932,
11933,11937,11941, 11942, 11946,11947,11948,11949, 11950, 11951, 11952, 11955,
11959,
11968,11972,11976, 11978, 11979,11980,11981,11986,11988,11989,
11991,11992,11995,
11997,11998, 12001,12002, 12004,12005,11899, 11905, 11910, 11916,11919,
11923,11924,
11925, 11928, 11929, 11931, 11934, 11938, 11943, 11953, 11954, 11956, 11960,
11963, 11964,
11967,11969,11973,11977,11982,11985,11987,11990,11993,11994,
11996,11999,12000,
12003, 12006, 12011, 12015, 12016, 12017, 12021, 12022, 12023, 12024, 12025,
12026, 12027,
12031,12034, 12042, 12043, 12045,12049,12050, 12051,12054, 12062, 12063,
12064, 12066,
12067, 12068, 12070, 12072, 12076, 12082, 12085, 12086, 12089, 12090, 12093,
12094, 12095,
12096,12097, 12098, 12101, 12103, 12107, 12108, 12111, 12112, 12114, 12118,
12119, 12120,
12128, 12007, 12010, 12012, 12020, 12028, 12032, 1203 3, 12035, 12038, 12039,
12044, 12046,
12052, 12055, 12056, 12057, 12058, 12059, 12060, 12061, 12065, 12069, 12071,
12073, 12077,
12080, 12081, 12084, 12087, 12088, 12091, 12099, 12100, 12102, 12104, 12109,
12110, 12113,
12115, 12121, 12122, 12123, 12124, 12125, 12127, 12129, 12130, 12135, 12136,
12141, 12144,
12147, 12148, 12149, 12153, 12159, 12160, 12131, 12134, 12137, 12138, 12139,
12140, 12142,
12143, 12145, 12146, 12150, 12154, 12157, 12158, 12161, 12162, 12163, 12164,
12168, 12171,
12176,12181, 12187,12190,12194,12196,12197,12199, 12201, 12202,12209, 12213,
12216,
12220,12221, 12223,12225, 12226,12231,12233, 12239, 12243, 12244, 12250,
12254, 12256,
12257, 12259, 12266, 12270, 12271, 12272, 12274, 12276, 12277, 12278, 12280,
12284, 12285,
12288, 12289, 12290, 12291, 12295, 12296, 12302, 12308, 12313, 12317, 12321,
12322, 12323,
CA 02528853 2005-12-23
12324, 12325, 12327, 12328, 12330, 12331, 12332, 12336, 12337, 12339, 12343,
12346, 12349,
12353, 12354, 12355, 12359, 12361, 12363, 123 67, 12369, 12370, 12372,
12376,12377, 12379,
12380, 12381, 12382, 123 83, 12385, 12389, 12393, 12398, 12400, 12402, 12403,
12406, 12408,
12409, 12410, 12412, 12415, 12417, 12419, 12422, 12424, 12429, 1243 0, 12432,
12433, 12435,
12436, 12437, 12441, 12445, 12448, 12449, 12454, 12458, 12459, 12460, 12165,
12169, 12170,
12172, 12175, 12177, 12180, 12182, 12185, 12186, 12188, 12191, 12195, 12198,
12200, 12203,
12206, 12207,12208, 12210, 12214,12217, 12222, 12224, 12227, 12228, 12229,
12230, 12232,
12234,12237,12238, 12240,12241,12242,12245,12248,12249, 12251,12255,12258,
12260,
12262, 12263, 12264, 12265, 12267, 12268, 12269, 12273, 12279, 12281, 12286,
12287, 12292,
12297, 12300, 12303, 12306, 12307, 12309, 12312, 12314, 12318, 12326, 12333,
12338, 12340,
12341, 12342, 12345, 12347, 12348, 12352, 12356, 12360, 12362, 12364, 12371,
12373, 12378,
12386,12390,12394, 12397, 12401, 12404, 12405,12411, 12413, 12418, 12420,
12423, 12425,
12428, 12438, 12439, 12440, 12442, 12443, 12446,12450, 12453, 12455, 12461,
12465, 12467,
12468, 12472,12474,12477,12480,12484,12486,12488, 12489, 12490, 12493, 12495,
12499,
12501, 12504, 12506, 12462, 12469, 12475, 12478, 12481, 12483, 12485, 12487,
12492, 12494,
12496, 12497, 12498, 12500,12502, 12505, 12507, 12511, 12517, 12520, 12522,
12523, 12524,
12528, 12530, 12534, 12535, 12536, 12537, 12541, 12542, 12547, 12550, 12551,
12552, 12554,
12555,12560,12564, 12565, 12569,12571,12574, 12579, 12581, 12586, 12590,
12592, 12593,
12594, 12596, 12600, 12601, 12602, 12604, 12610, 12613, 12614, 12615, 12616,
12620, 12626,
12630, 12508, 12512, 12515, 12518, 12521, 12525, 12529, 12531, 12538, 12543,
12546, 12548,
12549, 12553, 12556, 12559, 12561, 12566, 12570, 12572, 12573, 12575, 12578,
12582, 12585,
12587, 12595, 12597, 12599, 12603, 12605, 12608, 12609, 12611, 12617, 12618,
12621, 12624,
12625, 12627,12631, 12636,12637,12638,12639,12643,12647, 12648, 12652, 12654,
12660,
12664, 12666, 12670, 12672, 12675, 12677, 12680, 12686, 12691, 12692, 12695,
12699, 12700,
2S 12701, 12703, 12705, 12709, 12710, 12714, 12715, 12716, 12718, 12719,
12720, 12724, 12725,
12728, 12732,12738, 12739,12743, 12744,12745,12747, 12748, 12752, 12757,
12763, 12764,
12766,12767, 12768, 12771,12776,12777,12781,12785,
12787,12788,12791,12792,12796,
12797, 12800, 12804, 12808, 12813, 12815, 12816, 12817, 12822, 12825, 12828,
12831, 12833,
12834, 12837,12839, 12841, 12842,12632, 12635,12640,12641,12644,12649,
12653,12655,
71
CA 02528853 2005-12-23
12658, 12661, 12662, 12663, 12665, 12667, 12671, 12673, 12678, 12679, 12681,
12682, 12683,
12684, 12685, 12687, 12688, 12689, 12690, 12693, 12694, 12696, 12702, 12704,
12706, 12712,
12713, 12717, 12721, 12726, 12729, 12730, 12731, 12733, 12736, 12737, 12740,
12746, 12749,
12750, 12751, 12753, 12754, 12755, 12756, 12758, 12760, 12761, 12762, 12765,
12769, 12770,
12772, 12775, 12778, 12782, 12786, 12789, 12790, 12793, 12794, 12795, 12798,
12799, 12801,
12805, 12809, 12812, 12814, 12818, 12821, 12823, 12824, 12826, 12827, 12832,
12835, 12836,
12838, 12840, 12843, 12844, 12845, 12849, 12850, 12854, 12857, 12862, 12866,
12867, 12868,
12870, 12872, 12880, 12881, 12884,12885, 12889, 12890, 12892, 12896, 12898,
12899, 12903,
12907, 12910, 12913, 12919, 12920, 12921, 12923, 12928, 12932, 12938, 12942,
12945, 12948,
12950, 12951, 12953, 12959, 12846, 12851, 12855, 1285 8, 12861, 12863, 12869,
12871, 12873,
12876, 12877, 12878, 12879, 12882, 12883, 12886, 12887, 12888, 12891, 12893,
12894, 12895,
12900, 12901, 12902, 12904, 12908, 12909, 12911, 12912, 12914, 12917, 12918,
12924, 12927,
12929, 12933,12936;12937, 12939, 12943, 12949, 12954, 12957, 12958, 12960,
12961, 12965,
12969, 12975, 12977, 12962, 12966, 12967, 12968, 12970, 12971, 12972, 12973,
12974, 12976,
12978, 12981, 12982, 12986, 12989, 12991, 12992, 12993, 12995, 12999, 13001,
12983, 12987,
12996, 12997, 12998, 13000, 13002, 13005, 13009, 13013, 13017, 13019, 13025,
13029, 13030,
13032, 13033, 13034, 13037, 13040, 13042, 13043, 13044, 13048, 13049, 13051,
13052, 13053,
13057, 13062, 13064, 13065, 13066, 13067, 13072, 13073, 13076, 13078, 13082,
13083, 13085,
13087, 13088, 13089, 13092, 13096, 13100, 13101, 13102, 13105, 13108, 13109,
13110, 13114,
13117, 13118, 13124, 13129, 13131,13132, 13138, 13144, 13146, 13147,
13148,13152, 13156,
13159, 13164, 13167, 13168, 13170,13171, 13172, 13173, 13174, 13184, 13185,
13006, 13010,-
13014, 13018, 13020, 13021, 13022,13023, 13024,13026,13031, 13035, 13036,
13038, 13039,
13041, 13045, 13050, 13054, 13058, 13061, 13063, 13068, 13069, 13070, 13071,
13074, 13075,
13077, 13079, 13084, 13086, 13090,13091, 13093, 13097, 13104, 13107, 13111,
13115, 13119,
13121, 13122, 13123, 13125, 13126, 13127, 13128, 13130, 13133, 13137, 13139,
13142, 13143,
13145, 13149, 13153, 13157, 13158, 13160, 13163, 13165, 13166, 13169, 13175,
13176, 13177,
13178, 13179, 13180, 13181, 13182, 13183, 13186, 13189, 13190, 13191, 13195,
13196, 13197,
13199, 13200, 13204, 13206, 13192, 13198, 13201, 13207, 13212, 13213, 13226,
13228, 13231,
13235, 13240, 13245, 13247, 13248, 13250, 13254,13256, 13258, 13264, 13265,
13268, 13269,
72
CA 02528853 2005-12-23
13273, 13274, 13277, 13279, 13280, 13281, 13286, 13208, 13211, 13214, 13217,
13220, 13222,
13224, 13225, 13227, 13229, 13232, 13233, 13234, 13237, 13239, 13241, 13242,
13244, 13246,
13249, 13251, 13255, 13257, 13259, 13262, 13263, 13266, 13267, 13270, 13275,
13276, 13282,
13285, 13641, 13645, 13646, 13649, 13650, 13652, 13653, 13654, 13655, 13658,
13659, 13642,
13647, 13648, 13656, 13660, 13661, 13664, 13669, 13670, 13671, 13672, 13673,
13675, 13676,
13677, 13681, 13683, 13685,13687,13689, 13691, 13693, 13696, 13665, 13668,
13674, 13678,
13682, 13684,13686,13688,13690,13692,13694, i3695,13697,
13701,13702,13703,13709,
13713,13714, 13715, 13719, 13721, 13722,13726,13728, 13732, 13735,
13740,13698, 13704,
13705, 13706, 13707, 13708, 13710,13716, 13717, 13718, 13720, 13723,
13724,13725, 13727,
13729, 13733, 13734, 13736, 13739,13741, 13744, 13745, 13749, 13751, 13752,
13753, 13754,
13758,13762,13765, 13766, 13768,13772, 13773,13774,13777, 13778, 13779, 13783,
13785,
13790, 13791,13797, 13802, 13803,13805, 13806,13807, 13809, 13812,
13814,13816, 13817,
13818, 13819,13820, 13823, 13824,13826, i3828,13746, 13755, 13759, 13763,
13764, 13767,
13769, 13775, 13776, 13780, 13784, 13786, 13788,13789,13792,13795,
13796,13798, 13800,
13804, 13808, 13813, 13815, 13821,13822, 13829, 13832, 13837, 13841,
13844,13845, 13846,
13849, 13854, 13855, 13856, 13857,13861,13865, 13869, 13871, 13875,
13879,13880,13833,
13836, 13838,13843, 13847, 13850,13853, 13858, 13862, 13866, 13870,
13872,13876, 13881,
13882, 13883,13889, 13894, 13902,13903, 13910, 13911, 13914, 13915,
13916,13928,13929,
13930, 13931, 13935, 13936, 13937,13944, 13947, 13948, 13950, 13960,
13961,13962, 13964,
13965,13966, 13968, 13970, 13971,13972,13976,13978, 13884, 13887, 13890,13893,
13895,
13898, 13900, 13904,13905, 13906, 13907,13908,13909,13912, 13913, 13917,
13920, 13921,
13922,13923,13924, 13925, 13926, 13932, 13938,13941,13942, 13943, 13945,13946,
13949,
13953, 13954,13956, 13957, 13958,13959, 13963,13967, 13969,13974, 13975,
13977,13980,
13981,13988, 13989, 13990, 13992, 13994,13995,13999, 13982, 13985, 13986,
13987, 13991,
13993, 13996, 14000,14001, 14002, 14003, 14004,14009, 14014,14017, 14020,
14024, 14026,
14029,14035,14036, 14040, 14043, 14045, 14051,14057, 14058, 14059, 14063,
14065, 14066,
14067,14068,14069,14070,14071,14073, 14074, 14078,14079, 14080, 14081, 14082,
14083,
14084,14085,14087, 14094, 14095, 14100, 14108,14109, 14113,14114,14118, 14120,
14121,
14122, 14130, 14133, 14138, 14141, 14142, 14143,14147, 14005, 14008, 14010,
14013, 14015,
73
CA 02528853 2005-12-23
14018, 14021, 14025, 14027, 14028,14030, 14033, 14034,14037,
14038,14039,14041, 14042,
14044, 14046,14049, 14050, 14052, 14055, 14056, 14060, 14064,
14072,14077,14086, 14088,
14089, 14090, 14091, 14092, 14093, 14096, 14097, 14098, 14099, 14101, 14102,
14103, 14104,
14105, 14106, 14107, 14110, 14115, 14123, 14126, 14127, 14132, 14134, 1413 5,
14136, 1413 7,
14139, 14140, 14144, 14145, 14148, 14151, 14155, 14157, 14161, 14166, 14171,
14175, 14183,
14187, 14190, 14192, 14194, 14195, 14196, 14200, 14201, 14152, 14156, 1415 8,
14162, 14165,
14167, 14170, 14172, 14176, 14179, 14180, 14182, 14184, 14185, 14186, 14188,
14189, 1419 1,
14193, 14197, 14198, 14199, 14202, 14203, 14204, 14210, 14211, 14216, 14217,
14223, 14226,
14228, 14229, 14230, 14233, 14235,14239, 14241, 14243, 14244, 14250, 14254,
14255, 14256,
14257, 14260, 14261, 14262, 14267, 14268, 14269, 14273, 14274,
14277,14281,14284, 14286,
14289, 14292, 14293, 14297, 14299, 14301, 14304, 14305, 14306, 14312, 14316,
14320, 14322,
14324, 14328, 14329, 14331, 14337, 14342, 14344, 14346, 14347, 14349, 14350,
14353, 14356,
14361, 14363, 14364, 14365, 14366, 14205, 14208, 14212, 14215, 14218, 14221,
14224, 14225,
14227, 14231,14236, 14240, 14242, 14245, 14246, 14247, 14251, 14263, 14266,
14270, 14278,.
14282, 14283, 14285, 14287, 14290, 14291, 14294, 14298, 14300, 14302, 14303,
14307, 14310,
14313, 14317, 14323, 14325, 14327, 14330, 14334, 14338, 14340, 14341, 14345,
14348, 14351,.
14352, 14354, 14355, 14357, 14358, 14359, 14360, 14367, 14368, 14372, 14373,
14375, 14386,
14369, 14374, 14376, 14379, 14380, 14381, 14382, 14383, 14384 and SEQ ID
NO:14385.
A fiuther preferred embodiment of the method comprises the use of blocker
oligonucleotides. T he u se o f s uch b locker o ligonucleotides h as b een d
escribed b y Yu e t a 1.,
BioTechniques 23:714-720, 1997. Blocldng probe oligonucleotides are hybridized
to the
bisulfite treated nucleic acid concurrently with the PCR primers. PCR
amplification of the
nucleic acid is terminated at the 5' position of the blocking probe, such that
amplification of a
nucleic acid is suppressed where the complementary sequence to the blocking
probe is present.
The probes may be designed to hybridize to the bisulfite treated nucleic acid
in a methylation
status specific manner. For example, for detection of methylated nucleic acids
within a
population of unmethylated nucleic acids, sappression of the amplification of
nucleic acids
which are unnaethylated at the position in question would be carried out by
the use of blocking
74
CA 02528853 2005-12-23
probes comprising a'CpA' or 'TpA' at the position in question, as opposed to
a'CpG' if the
suppression of amplification of methylated nucleic acids is desired.
For PCR methods using blocker oligonucleotides, efficient disruption of
polymerase-
mediated amplification requires that blocker oligonucleotides not be elongated
by the
polymerase. Preferably, this is achieved through the use of blockers that are
3'-
deoxyoligonucleotides, or oligonucleotides derivitized at the 3' position with
other than a"free"
hydroxyl group. For example, 3'-O-acetyl oligonucleotides are representative
of a preferred
class of blocker molecule.
Additionally, polymerase-mediated decomposition of the blocker
oligonucleotides
should be precluded. Preferably, such preclusion comprises either use of a
polymerase lacking
5'-3' exonuclease activity, or use of modified blocker oligonucleotides
having, for example,
thioate bridges at the 5'-tenminii thereof that render the blocker molecule
nuclease-resistant.
Particular applications may not require such 5' modifications of the blocker.
For example, if the
blocker- and primer-binding sites overlap, lhereby precluding binding of the
primer (e.g., with
excess blocker), degradation of the blocker oligonucleotide will be
substantially precluded. This
is because the polymerase will not extend the primer toward, and through (in
the 5'-3' direction)
the blocker-a process that normally results in degradation of the hybridized
blocker
oligonucleotide.
A particularly preferred blocker/PCR embodiment, for purposes of the present
invention
and as implemented herein, comprises the use of peptide nucleic acid (PNA)
oligomers as
blocking o ligonucleotides. S uch PNA b locker o ligomers are i deally s
uited, b ecause they are
neither decomposed nor extended by the polymerase.
Preferably, therefore, the base sequence of said blocking oligonucleotides is
required to
comprise a sequence having a length of at least 9 nucleotides which hybridizes
to a treated
nucleic acid sequence according to one of SEQ ID NOS:304 to SEQ ID NO:535 and
sequences
complementary thereto, wherein the base sequence of said oligonucleotides
comprises at least
one CpG, TpG or CpA dinucleotide.
in a further preferred embodiment of the method, the blocking oligonucleotides
are
selected from the group consisting of SEQ ID NOS:2114, 2119, 2124, 2125, 2127,
2132, 2133,
CA 02528853 2005-12-23
2135, 2137, 2138, 2139, 2140, 2141, 2142, 2143, 2144, 2146, 2147, 2148, 2149,
2150, 2151,
2152, 2153, 2154, 2155, 2156, 2157, 2158, 2159, 2160, 2031, 2082, 2087, 2089,
2094, 2095,
2096, 2097, 2099, 2107, 1980, 2018, 2022, 2023, 2026, 1939, 1966, 1973, 1974,
1975, 1926,
1930, 1931, 1932, 1933, 1934, 1903, 1917, 1921, 1834, 1896, 1805, 1819, 1821,
1823, 1824,
1825, 1826, 1827, 1828, 1829, 1770, 1782, 1785, 1786, 1788, 1789, 1790, 1792,
1795, 1796,
1797, 1798, 1799, 1800, 1748, 1759, 1760, 1761, 1762, 1763, 1764, 1765, 1724,
1741, 1597,
1609, 1610, 1611, 1612, 1613, 1614, 1615, 1616, 1617, 1618, 1619, 1620, 1621,
1622, 1623,
1624, 1625, 1626, 1627, 1628, 1629, 1630, 1631, 1633, 1634, 1635, 1636, 1637,
1638, 1639,
1640, 1641, 1642, 1643, 1644, 1645, 1646, 1647, 1648, 1649, 1650, 1651, 1652,
1653, 1654,
1655, 1656, 1657, 1658, 1659, 1660, 1661, 1662, 1663, 1668, 1684, 1685, 1686,
1687, 1688,
1689, 1690, 1691, 1692, 1693, 1694, 1695, 1696, 1697, 1698, 1699, 1700, 1701,
1706, 1712,
1713, 1715, 1716, 1717, 1718, 1719, 1571, 1579, 1581, 1584, 1585, 1586, 1587,
1588, 1591,
1592, 1537, 1555, 1556, 1557, 1558, 1559, 1560, 1561, 1562, 1563, 1564, 1565,
1566, 1452,
1455, 1456, 1457, 1458, 1463, 1514, 1515, 1516, 1517, 1518, 1519, 1524, 1525,
1526, 1527,
1528, 1529, 1531, 1532, 1372, 1423, 1424, 1425, 1427, 1432, 1433, 1434, 1435,
1436, 1437,
1440, 1441, 1442, 1443, 1444, 1445, 1446, 1447, 1343, 1358, 1359, 1360, 1361,
1362, 1363,
1364, 1365, 1366, 1367, 1310, 1315, 1316, 1317, 1318, 1319, 1320, 1321, 1322,
1324, 1325,
1327, 1328, 1330, 1332, 1334, 1337, 1338, 1276, 1290, 1305, 1203, 1228, 1238,
1239, 1240,
1247, 1248, 1255, 1256, 1265, 1268, 1269, 1164, 1171, 1172, 1173, 1174, 1176,
1177, 1178,
1179, 1180, 1181, 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189; 1190, 1191,
1192, 1193,
1194, 1195, 1196, 1197, 1198, 2725, 2748, 2749, 2750, 2710, 2717, 2718, 2719,
2720, 2721,
2684, 2699, 2703, 2704, 2705, 2706, 2658, 2662, 2679, 2603, 2613, 2617, 2585,
2594, 2595,
2596, 2597, 2598, 2599, 2489, 2497, 2498, 2499, 2500, 2501, 2504, 2505, 2507,
2508, 2310,
2511, 2512, 2513, 2514, 2515, 2519, 2520, 2521, 2522, 2525, 2526, 2527, 2528,
2529, 2530,
2531, 2532, 2534, 2535, 2536, 2537, 2539, 2540, 2541, 2542, 2543, 2544, 2545,
2546, 2547,
2550, 2551, 2552, 2553, 2554, 2558, 2563,, 2564, 2572, 2580, 2581, 2472, 2482,
2484, 2408,
2464, 2465, 2466, 2467, 2468, 2339, 2349, 2367, 2370, 2372, 2373, 2374, 2375,
2376, 2377,
2378, 2379, 2380, 2382, 2386, 2387, 2389, 2393, 2394, 2395, 2396, 2397, 2398,
2402, 2403,
2404, 2327, 2333, 2334, 2335, 2305, 2315, 2318, 2260, 2268, 2270, 2271, 2272,
2273, 2274,
76
CA 02528853 2005-12-23
2275, 2276, 2277, 2278, 2279, 2280, 2281, 2282, 2283, 2284, 2285, 2286, 2287,
2288, 2289,
2290, 2291, 2292, 2293, 2294, 2295, 2296, 2297, 2298, 2299, 2300, 2301, 2232,
2245, 2246,
2248, 2249, 2250, 2251, 2252, 2254, 2255, 2256, 2210, 2218, 2219, 2221, 2222,
2223, 2227,
2228, 2202, 2204, 2205, 2206, 2164, 2180, 2183, 2187, 2188, 2189, 2190, 2191,
2192, 2193,
2194, 2195, 2196, 2197 and 2198.
The fragments obtained by means of the amplification can carry a direetly or
indirectly
detectable label. Preferred are labels in the form of fluorescence labels,
radionuclides, or
detachable molecule fragments having a typical mass which can be detected in a
mass
specftmeter. W here s aid 1 abels are m ass 1 abels, i t i s p referred t hat
the 1 abeled arnplificates
have a single positive or negative net charge, allowing for better
detectability in the mass
spectrometer. The detection may be carried out and visualized by means o4
e.g., matrix assisted
laser desorptionlionization mass spectrometry (MALDl) or using electron spray
mass
vwftmetrv (ESI).
1Vlatrix Assisted Laser Desorption/Ionization Mass Spectrometry (MALDI TOF) is
a
very efficient development for the analysis of biomolecules (Karas &
Hilienkamp, Anal Chem.,.
60:2299-301, 1988). An analyte is embedded in a light-absorbing matrix. The
matrix isi
evaporated by a sb.ort laser pulse thus transporting the analyte molecule into
the vapour phase in
an unfragmented manner. The analyte is ionized by collisions with matrix
molecules. An
applied voltage accelerates the ions into a field-free flight tube. Due to
their different masses,
the ions are accelerated at different rates. - Smaller ions reach the detector
sooner than bigger
ones. MALDI-TOF spectrometry is well suited to the analysis of peptides and
proteins. The
analysis of nucleic acids is somewhat more difficult (Gut & Beck, Current
Innovations and
F'uture Trends, 1:147-57, 1995). The sensitivity with respect to nucleic acid
analysis is
approximately 100-times less than for peptides, and decreases
disproportionally with increasing
fragment size. Moreover, for nucleic acids having a multiply negatively
charged backbone, the
ionization process via the matrix is considerably less efficient. 7n MALDI-TOF
spectrometry,
the selection of the matrix plays an eminently important role. For desorption
of peptides, several
very efficient matrixes have been found which produce a very fine
erystallisation. There are
now several responsive matrixes for DNA, however, the difference in
sensitivity between
77
CA 02528853 2005-12-23
peptides aad nucleic acids has not been reduced. This difference in
sensitivity can be reduced,
however, by chemically modifying the DNA in such a manner that it becomes more
similar to a
peptide. For example, phosphorothioate nucleic acids, in which the usual
phosphates of the
backbone are sabstituted with thiophosphates, can be converted into a charge-
neutral DNA using
simple alkylation chemistry (Gut & Beck, Nucleic Acids Res. 23: 1367-73,
1995). The coupling
of a charge tag to this modified DNA results in an increase in MALDI-TOF
sensitivity to the
same level as that found for peptides. A further advantage of charge tagging
is the increased
stability of the analysis against impurities, which makes the detection of
unmodifsed substrates
conaiderably more difficult.
In the fourth siep of the method, the amplificates obtained during the third
step of the
method are analysed in order to ascertain the methylation statas of the CpG
dinueleotides prior
to the treatmeart.
In embodiments where the amplificates were obtained by means of MSP
amplification,
the presence or absence of an ampliftcate is in itself indicative of the
methylation state of the
CpG positions covered by the primer, according to the base sequences of said
primer.
Atnplificates obtained by ineans of both standard and methylation specific PCR
may be
ftuther analyzed by means of hybridization-based methods such as, but not
limited to, atray
technology and probe based technologies as well as by means of teclrniques
such as sequeneing
and template directed extension.
In one embodiment of the method, the amplificates synthesised in step three
are
subsequently h ybridized to an a xray or a set o f o ligonucleotides and/or
PNA probes. In t his
context, the hybridization takes place in the following manuer: the set of
probes used during the
hybridization is preferably composed of at least 2 oligonucleotides or PNA-
oligomers; in the
process, the amplificates serve as probes which hybridize to oligonucleotides
previously bonded
to a solid phase; the non-hybridized fragments are subsequently removed; said
oligonucleotides
contain at least one b ase sequence h aving a 1 ength oÃat 1 east 9
nucleotides which i s reverse
complementary or identical to a segment of the base sequences specified in the
present Sequence
Listing; andd the segment comprises at least one CpG, TpG or CpA dinucleotide.
In a preferred embodiment, said dinucleotide is present in the central third
of the
78
CA 02528853 2005-12-23
oligomer. For example, wherein the oligomer comprises one CpG dinucleotide,
said
dinucleotide is preferably the fifth to ninth nucleotide from the 5'-end of a
13-mer. One
oligonucleotide exists for the analysis of each CpG dinucleotide within the
sequence according
to SEQ ID NOS:1 to SEQ ID NO:64, and the equivalent positions within SEQ ID
NOS:304 to
SEQ ID NO:535. Said oligonucleotides may also be present in the form of
peptide nucleic
acids. The non-hybridized amplificates are then removed. The hybridmed
amplificates are then
detected. In this context, it is preferred that labels attached to the
amplificates are identifiable at
each position of the solid phase at which an oligonucleotide sequence is
located.
In yet a fnrther embodiment of the method, the genomic methylation status of
the CpG
positions may be ascertained by means of oligonucleotide probes that are
bybridised to the
bisulfite treated DNA concurrently with the PCR amplification primers (wherein
said primers
may either be methylation specific or standard).
A particularly prefared embodiment of this method is the use of fluorescence-
based
Real Time Quantitative PCR (Iieid et al., Genome Res. 6:986-994, 1996; also
see United States
Patent No. 6,331,393) employing a dual-labeled fluorescent oligonucleotide
probe (TaqMan''M
PCR, using an ABI Prism 7700 Sequence Detection System, Perldn Elmer Applied
Biosystems,
Poster City, California). The TaqManTM PCR reaction employs the use of a
nonextendible
interrogating o ligonucleotide, c alled a T.aqManTu p robe, w hich, i n p
refeaed embodiments, i s-
designed to hybridi2e to a GpC-rich sequence located between the forward and
reverse
amplification primers. The TaqManTm probe ftuther comprises a fluorescent
"'reporter moiety"
and a"queneher moiety" covalently bound to linker nioieties (e.g.,
phosphoramidites) attached
to the nucleotides of the TaqMan'rM oligonucleotide. For analysis of
methylation within nucleie
acids subsequent to bisulfite treatment, it is required that the probe be
methylation specific, as
descn'bed in United States Patent No. 6,331,393, (hereby incorporated by
reference in its
entirety) also known as the MethylLightTu assay. Variations on the TasllvianTu
detection
methodology that are also suitable for use with the described invention
include the use of dual-
probe technology (LightcyelerT)A) or fluotescent amplification primers
(SunriseTm technology).
Both these techniques may be adapted in a manner suitable for use with
bisulfite treated DNA,
and moreover for methylation analysis within CpG dinucleotides.
79
CA 02528853 2005-12-23
A further s uitable m ethod for t he u se o f p robe o ligonucleotides for t
he assessment o f
methylation by analysis of bisulfite treated nucleic acids In a further
preferred embodiment of
the method, the,ffth step of the method comprises the use of template-directed
oligonucleotide
extension, such as MS-SNuPE as described by Gonzalgo & Jones, Nucleic Acids
Res. 25:2529-
2531,1997.
In yet a further embodiment of the method, the fifth step of the method
comprises
sequencing and subsequent sequence analysis of the amplificate generated in
the third step of
the method (Sanger F., et a1., Proc Natl Acad Sci USA 74:5463-5467, 1977).
B est Mode
In the most preferred embodiment of the method the genomic nucleic acids are
isolated
and treated according to the first three steps of the method outlined above,
namely:
a) obtaining, from a subject, a biological sample having subject genomic DNA;
b) extracting or otherwise isolating the genomic DNA;
c) treating the genomic DNA of b), or a fragment thereof, with one or more
reagents to convert cytosine bases that are unmethylated in the 5-position
thereof to uracil or to
another base that is detectably dissimilar to cytosine in terms of
hybridization properties; and
wherein
d) amplifying subsequent to treatment in c) is carried out in a methylation
specific
manner, namely by use of methylation specific primers or blocking
oligonucleotides, and
further wherein
e) detecting of the amplificates is carried out by means of a real-time
detection
probe, as described above.
Preferably, where the subsequent amplification of d) is carried out by means
of
meth}tlation specific primers, as descn'bed above, said methylation specific
primers comprise a
sequence having a length of at least 9 mbcleotides which hybridizes to a
treated nucleic acid
sequence according to one of SEQ ID NOS:304 to SEQ ID NO:535 and sequences
complementary t hereto, wherein t he b ase s equence o f sa id o ligomers
comprises a t 1 east o ne
CpG dinucleotide.
CA 02528853 2005-12-23
In a ftnther preferred embodiment of the method, the MSP primers are selected
from the
group consisting of SEQ ID NOS:2754, 2757, 2759, 2767, 2768, 2772, 2776, 2779,
2784,
2785, 2786, 2789, 2791, 2792, 2794, 2797, 2798, 2801, 2805, 2806, 2809, 2811,
2815, 2817,
2824, 2830, 2832, 2833, 2834, 2838, 2840, 2843, 2846, 2850, 2851, 2853, 2855,
2859, 2866,
2867, 2870, 2871, 2876, 2878, 2881, 2885, 2886, 2892, 2893, 2895, 2896, 2898,
2904, 2912,
2915, 2917, 2919, 2922, 2923, 2929, 2931, 2939, 2941, 2944, 2946, 2948, 2950,
2955, 2956,
2957, 2960, 2962, 2965, 2966, 2967, 2968, 2969, 2971, 2975, 2976, 2977, 2978,
2979, 2980,
2981, 2984, 2988, 2989, 2990, 2992, 2994, 2997, 2999, 3001, 3002, 3004, 3006,
3007, 3008,
3010, 3013, 3014, 3016, 3017, 3018, 3020, 3022, 3025, 2752, 2755, 2760, 2762,
2763, 2764,
2765, 2769, 2771, 2773, 2775, 2777, 2780, 2781, 2782, 2783, 2787, 2788, 2790,
2793, 2795,
2796, 2800, 2802, 2803, 2804, 2808, 2810, 2812, 2814, 2816, 2818, 2820, 2821,
2823, 2825,
2827, 2828, 2829, 2831, 2835, 2836, 2837, 2839, 2841, 2842, 2844, 2845, 2847,
2849, 2852,
2854, 2856, 2858, 2860, 2861, 2862, 2863, 2864, 2865, 2868, 2869, 2872, 2873,
2874, 2875,
2877, 2879, 2880, 2882, 2883, 2884, 2887, 2888, 2889, 2890, 2891, 2894, 2897,
2899, 2901,
2902, 2903, 2905, 2907, 2909, 2911, 2914, 2916, 2918, 2920, 2924, 2926, 2927,
2928, 2930,
2932, 2933, 2934, 2935, 2936, 2937, 2938, 2942, 2945, 2947, 2949, 2951, 2953,
2954, 2958,
2959, 2961, 2963, 2970, 2972, 2973, 2974, 2982, 2985, 2987, 2991, 2993, 2995,
2996, 2998,
3000, 3003, 3005, 3011, 3019, 3021, 3023, 3026, 3027, 3028, 3032, 3033, 3036,
3037, 3038,
3039, 3041, 3042, 3043, 3044, 3047, 3048, 3049, 3052, 3055, 3059, 3061, 3064,
3065, 3068,
3069, 3071, 3072, 3075, 3076, 3080, 3083, 3084, 3085, 3086, 3091, 3093, 3096,
3097, 3100,
3104, 3109, 3110, 3113, 3115, 3117, 3118, 3123, 3126, 3127, 3130, 3134, 3135,
3136, 3138,
3139, 3144, 3146, 3147, 3149, 3150, 3155, 3029, 3031, 3034, 3040, 3045, 3050,
3051, 3053,
3054, 3056, 3057, 3060, 3063, 3066, 3073, 3078, 3081, 3088, 3089, 3090, 3092,
3094, 3098,
3101, 3103, 3105, 3107, 3108, 3111, 3114, 3116, 3119, 3121, 3122, 3124, 3128,
3131, 3133,
3137, 3140, 3142, 3145, 3148, 3151, 3152, 3153, 3157, 3159, 3165, 3166, 3167,
3170, 3171,
3172, 3174, 3175, 3176, 3179, 3180, 3181, 3184, 3185, 3186, 3192, 3194, 3198,
3201, 3202,
3203, 3204, 3206, 3209, 3212, 3213, 3215, 3216, 3217, 3218, 3221, 3224, 3226,
3228, 3229,
3230, 3232, 3233, 3234, 3235, 3237, 3238, 3239, 3245, 3247, 3248, 3249, 3160,
3162, 3163,
3164, 3168, 3169, 3173, 3178, 3182, 3183, 3187, 3189, 3190, 3195, 3196, 3197,
3199, 3205,
81
CA 02528853 2005-12-23
3207, 3210, 3214, 3219, 3222, 3223, 3225, 3227, 3236, 3240, 3242, 3243, 3244,
3246, 3250,
3252, 3256, 3261, 3264, 3268, 3269, 3270, 3273, 3275, 3279, 3280, 3281, 3282,
3283, 3284,
3285, 3286, 3290, 3292, 3295, 3297, 3301, 3304, 3305, 3306, 3307, 3308, 3310,
3311, 3312,
3315, 3319, 3320, 3324, 3327, 3331, 3333, 3334, 3339, 3348, 3351, 3354, 3358,
3360, 3364,
3367, 3368, 3374, 3376, 3253, 3255, 3257, 3259-, 3260, 3262, 3265, 3266, 3267,
3271, 3274,
3276, 3278, 3287, 3289, 3291, 3293, 3298, 3300, 3302, 3309, 3313, 3314, 3316,
3318, 3321,
3322, 3325, 3328, 3330, 3332, 3335, 3337, 3338, 3340, 3342, 3343, 3344, 3345,
3346, 3347,
3349, 3350, 3352, 3353, 3355, 3356, 3357, 3359, 3361, 3363, 3365, 3369, 3371,
3372, 3373,
3377, 3380, 3383, 3386, 3388, 3389, 3390, 3391, 3392, 3395, 3396, 3400, 3401,
3403, 3406,
3408, 3413, 3414, 3417, 3420, 3423, 3428, 3432, 3433, 3434, 3436, 3439, 3440,
3442, 3443,
3445, 3447, 3453, 3455, 3458, 3463, 3466, 3469, 3470, 3471, 3472, 3473, 3474,
3475, 3477,
3478, 3480, 3481, 3483, 3484, 3487, 3490, 3494, 3495, 3496, 3497, 3499, 3500,
3504, 3505,
3508, 3512, 3514, 3515, 3516, 3521, 3525, 3529, 3533, 3534, 3537, 3538, 3540,
3541, 3542,
3543, 3544, 3547, 3548, 3553, 3557, 3558, 3559, 3560, 3562, 3563, 3566, 3571,
3574, 3575,
3577, 3580, 3583, 3584, 3585, 3586, 3592, 3594, 3595, 3597, 3598, 3599, 3601,
3603, 3604,
3607, 3608, 3612, 3619, 3621, 3622, 3626, 3631, 3632, 3637, 3638, 3639, 3640,
3642, 3643,.
3644, 3646, 3647, 3649, 3650, 3652, 3656, 3657, 3660, 3663, 3668, 3670, 3673,
3676, 3677,
3678, 3683, 3688, 3690, 3694, 3697, 3700, 3702, 3703, 3704, 3708, 3404, 3409,
3411, 3415,
3418, 3419, 3421, 3424, 3426, 3427, 3429, 3431, 3435, 3437, 3438, 3441, 3444,
3446, 3448,
3450, 3452, 3454, 3456, 3459, 3461, 3462, 3464, 3467, 3476, 3479, 3482, 3485,
3488, 3489,
3491, 3492, 3493, 3501, 3503, 3506, 3509, 3510, 3511, 3513, 3517, 3518, 3519,
3520, 3522,
3524, 3526, 3527, 3528, 3530, 3531, 3535, 3536, 3545, 3549, 3551, 3552, 3554,
3555, 3556,
3561, 3564, 3565, 3568, 3569, 3570, 3573, 3576, 3578, 3581, 3587, 3588, 3589,
3590, 3591,
3593, 3596, 3600, 3602, 3605, 3606, 3609, 3611, 3613, 3614, 3615, 3616, 3617,
3618, 3623,
3624, 3625, 3627, 3629, 3630, 3633, 3634, 3635, 3636, 3641, 3645, 3651, 3653,
3654, 3655,
3658, 3661, 3662, 3665, 3666, 3667, 3669, 3671, 3674, 3679, 3681, 3684, 3686,
3689, 3691,
3692, 3693, 3695, 3696, 3698, 3701, 3705, 3706, 3707, 3709, 3710, 3714, 3715,
3717, 3718,
3722, 3723, 3729, 3730, 3732, 3735, 3739, 3740, 3741, 3743, 3744, 3745, 3746,
3749, 3753,
3754, 3755, 3756, 3711, 3713, 3719, 3721, 3724, 3725, 3726, 3727, 3728, 3731,
3733, 3736,
82
CA 02528853 2005-12-23
3737, 3738, 3742, 3747, 3748, 3750, 3752, 3757, 3758, 3759, 3760, 3761, 3764,
3767, 3771,
3773, 3775, 3776, 3777, 3781, 3785, 3786, 3787, 3790, 3791, 3793, 3796, 3797,
3798, 3799,
3805, 3809, 3814, 3817, 3818, 3822, 3823, 3828, 3833, 3836, 3844, 3847, 3850,
3851, 3853,
3856, 3857, 3862, 3873, 3876, 3877, 3880, 3885, 3887, 3889, 3890, 3892, 3893,
3895, 3896,
3899, 3902, 3903, 3905, 3906, 3907, 3908, 3916, 3923, 3926, 3927, 3930, 3935,
3936, 3938,
3939, 3940, 3944, 3945, 3949, 3950, 3951, 3954, 3955, 3957, 3960, 3965, 3970,
3971, 3978,
3979, 3982, 3983, 3984, 3985, 3762, 3765, 3768, 3770, 3772, 3774, 3778, 3779,
3780, 3782,
3784, 3788, 3792, 3794, 3800, 3801, 3802, 3803, 3804, 3806, 3807, 3808, 3810,
3812, 3813,
3815, 3819, 3820, 3821, 3824, 3826, 3827, 3829, 3831, 3832, 3834, 383?, 3838,
3839, 3840,
3842, 3845, 3848, 3852, 3854, 3858, 3860, 3861, 3863, 3864, 3865, 3866, 3867,
3868, 3869,
3870, 3871, 3872, 3874, 3875, 3878, 3881, 3883, 3884, 3886, 3888, 3891, 3894,
3897, 3898,
3900, 3901, 3904, 3909, 3911, 3914, 3915, 3917, 3919, 3920, 3921, 3922, 3924,
3928, 3931,
3933, 3934, 3937, 3941, 3942, 3943, 3947, 3948, 3952, 3958, 3959, 3961, 3962,
3963, 3964,
3966, 3968, 3972, 3974, 3975, 3976, 3980, 3981, 3986, 3987, 3990, 3993, 3994,
3997, 4000,
4003, 4004, 4006, 4012, 4015, 4017, 4020, 4024, 4027, 4029, 4031, 4032, 4033;
4037, 4038,
4040, 4042, 4047, 4048, 4050, 4051, 4052, 4054, 4055, 4058, 4063, 4066, 4069,
4070, 4072,
4073, 4076, 4078, 4079, 4082, 4083, 4084, 4085, 4086, 4090, 4093, 4094, 4095,
4096, 4099,
4100, 4104, 4106, 4113, 4116, 4119, 4120, 4124, 4127, 4130, 4134, 4135, 4136,
4138, 4142,
4145, 4146, 4149, 4150, 4152, 4153, 4159, 4160, 4161, 4163, 4168, 4169, 4171,
4173, 4176,
4180, 4181, 4185, 4189, 4191, 4192, 4194, 4197, 4198, 4199, 4201, 4206, 4207,
4208, 4213,
4219, 4220, 4223, 4224, 4225, 4228, 4231, 4232, 4233, 4237, 4238, 4242, 4243,
4246, 4247,
4251, 4254, 4255, 4256, 4258, 3988, 3991, 3995, 3998, 4001, 4005, 4007, 4009,
4010, 4011,
4013, 4016, 4018, 4019, 4021, 4022, 4023, 4025, 4028, 4034, 4035, 4039, 4043,
4045, 4046,
4049, 4056, 4059, 4061, 4064, 4065, 4067, 4068, 4071, 4074, 4075, 4077, 4080,
4087, 4088,
4089, 4092, 4098, 4101, 4102, 4105, 4108, 4109, 4110, 4111, 4112, 4114, 4115,
4117, 4121,
4122, 4123, 4125, 4128, 4129, 4131, 4132, 4133, 4137, 4139, 4141, 4144, 4148,
4151, 4154,
4156, 4157, 4162, 4165, 4166, 4167, 4170, 4172, 4174, 4177, 4179, 4182, 4184,
4186, 4188,
4190, 4195, 4196, 4202, 4204, 4205, 4209, 4211, 4214, 4216, 4217, 4218, 4221,
4226, 4229,
4234, 4236, 4239, 4241, 4244, 4245, 4248, 4249, 4252, 4257, 4259, 4260, 4261,
4262, 4266,
83
CA 02528853 2005-12-23
4267, 4268, 4271, 4272, 4275, 4276, 4279, 4281, 4282, 4284, 4285, 4287, 4289,
4290, 4292,
4295, 4297, 4299, 4301, 4306, 4310, 4316, 4317, 4318, 4321, 4323, 4325, 4328,
4334, 4335,
4337, 4341, 4342, 4343, 4344, 4348, 4350, 4351, 4352, 4354, 4357, 4358, 4359,
4364, 4365,
4366, 4370, 4371, 4372, 4373, 4376, 4378, 4381, 4383, 4384, 4386, 4388, 4396,
4397, 4399,
4400, 4403, 4405, 4407, 4409, 4412, 4415, 4416, 4417, 4424, 4426, 4429, 4433,
4435, 4437,
4442, 4443, 4447, 4450, 4451, 4452, 4455, 4457, 4460, 4461, 4462, 4465, 4466,
4469, 4470,
4474, 4478, 4481, 4484, 4488, 4491, 4494, 4495, 4497, 4498, 4504, 4506, 4509,
4510, 4511,
4514, 4516, 4518, 4520, 4521, 4523, 4525, 4528, 4530, 4533, 4534, 4535, 4538,
4540, 4542,
4545, 4547, 4548, 4553, 4554, 4555, 4556, 4557, 4562, 4564, 4567, 4568, 4569,
4579, 4580,
4582, 4584, 4587, 4589, 4593, 4596, 4597, 4598, 4600, 4601, 4602, 4605, 4606,
4607, 4609,
4610, 4613, 4614, 4616, 4618, 4621, 4624, 4626, 4627, 4628, 4631, 4632, 4636,
4639, 4641,
4644, 4645, 4648, 4263, 4265, 4269, 4270, 4273, 4274, 4277, 4280, 4283, 4286,
4288, 4291,
4293, 4296, 4298, 4300, 4302, 4303, 4304, 4305, 4307, 4308, 4309, 4311, 4313,
4315, 4319,
4322, 4324, 4326, 4327, 4330, 4331, 4332, 4333, 4336, 4338, 4340, 4345, 4347,
4349, 4353,
4355, 4360, 4361, 4362, 4367, 4368, 4369, 4374, 4375, 4377, 4379, 4382, 4385,
4387, 4389,
4390, 4391, 4392, 4393, 4394, 4395, 4398, 4401, 4406, 4408, 4410, 4411, 4413,
4418, 4420,.
4422, 4423, 4425, 4427, 4428, 4430, 4432, 4434, 4436, 4438, 4440, 4441, 4444,
4446, 4448;
4453, 4454, 4456, 4458, 4463, 4464, 4467, 4471, 4472, 4473, 4475, 4476, 4477,
4479, 4480,
4482, 4485, 4486, 4487, 4489, 4490, 4492, 4496, 4499, 4501, 4502, 4503, 4505,
4507, 4508,
4512, 4515, 4517, 4519, 4522, 4524, 4529, 4531, 4536, 4537, 4539, 4541, 4543,
4546, 4549,
4551, 4558, 4559, 4560, 4561, 4563,4565, 4570, 4571, 4572, 4573, 4574, 4575,
4576, 4578,
4581, 4583, 4585, 4586, 4588, 4590, 4592, 4595, 4599, 4603, 4611, 4612, 4615,
4617, 4619,
4620, 4622, 4630, 4633, 4634, 4635, 4638, 4640, 4642, 4643, 4646, 4647, 4649,
4652, 4655,
4659, 4661, 4663, 4664, 4665, 4668, 4671, 4674, 4675, 4676, 4678, 4680, 4683,
4686, 4690,
4696, 4700, 4703, 4704, 4706, 4709, 4711, 4714, 4715, 4717, 4718, 4719, 4720,
4721, 4726,
4727, 4730, 4732, 4738, 4742, 4745, 4749, 4750, 4753, 4755, 4759, 4763, 4768,
4769, 4772,
4773, 4776, 4778, 4779, 4782, 4784, 4789, 4790, 4794, 4797, 4801, 4805, 4807,
4813, 4814,
4819, 4821, 4822, 4827, 4829, 4833, 4834, 4835, 4837, 4838, 4841, 4842, 4843,
4844, 4847,
4849, 4850, 4852, 4853, 4859, 4860, 4861, 4865, 4867, 4868, 4869, 4870, 4873,
4876, 4877,
84
CA 02528853 2005-12-23
3{j
4878, 4879, 4883, 4885, 4889, 4891, 4892, 4894, 4895, 4897, 4898, 4903, 4906,
4911, 4912,
4918, 4919, 4920, 4924, 4927, 4929, 4932, 4934, 4941, 4942, 4945, 4948, 4949,
4950, 4650,
4653, 4656, 4658, 4660, 4662, 4666, 4667, 4669, 4670, 4672, 4677, 4681, 4684,
4685, 4687,
4688, 4689, 4691, 4693, 4694, 4695, 4697, 4699, 4701, 4702, 4705, 4707, 4708,
4716, 4722,
4724, 4728, 4731, 4733, 4735, 4736, 4737, 4739, 4741, 4744, 4746, 4748, 4751,
4754, 4756,
4758, 4760, 4761, 4764, 4766, 4767, 4770, 4771, 4774, 4777, 4780, 4783, 4785,
4786, 4787,
4788, 4791, 4792, 4793, 4795, 4798, 4800, 4802, 4804, 4806, 4808, 4810, 4811,
4812, 4815,
4816, 4817, 4818, 4820, 4823, 4824, 4825, 4828, 4830, 4832, 4836, 4839, 4840,
4845, 4846,
4848, 4851, 4854, 4855, 4856, 4857, 4858, 4862, 4864, 4871, 4872, 4874, 4875,
4881, 4882,
4884, 4886, 4887, 4890, 4893, 4896, 4899, 4900, 4901, 4902, 4904, 4905, 4907,
4908, 4909,
4910, 4913, 4915, 4917, 4921, 4922, 4923, 4925, 4926, 4928, 4930, 4935, 4936,
4937, 4938,
4940, 4943, 4944, 4946, 4947, 4951, 4952, 4953, 4954, 4956, 4957, 4960, 4963,
4967, 4970,
4971, 4972, 4976, 4977, 4985, 4986, 4987, 4989, 4991, 4992, 4997, 4999, 5002,
5003, 5004,
5006, 5010, 5013, 5014, 5015, 5017, 5018, 5019, 5020, 5021, 5024, 5025, 5027,
5031, 5032,
5033, 5037, 5040, 5041, 5042, 5043, 4958, 4961, 4964, 4966, 4968, 4969, 4973,
4974, 4975,
4978, 4979, 4980, 4981, 4982, 4983, 4984, 4988, 4990, 4993, 4995, 4996, 4998,
5000, 5005,
5007, 5009, 5011, 5016, 5022, 5026, 5028, 5030, 5034, 5036, 5038, 5039, 5044,
5047, 5049,
5050, 5052, 5054, 5055, 5057, 5060, 5061, 5064, 5045, 5048, 5051, 5053, 5056,
5058, 5062,
5065, 5066, 5069, 5073, 5077, 5080, 5083, 5087, 5093, 5096, 5099, 5100, 5103,
5104, 5106,
5111, 5116, 5119, 5120, 5125, 5128, 5132, 5134, 5135, 5137, 5139, 5140, 5142,
5144, 5148,
5149, 5150, 5155, 5157, 5158, 5160, 5166, 5168, 5170, 5171, 5173, 5176, 5177,
5179, 5184,
5187, 5188, 5190, 5193, 5195, 5198, 5201, 5204, 5208, 5211, 5215, 5216, 5217,
5220, 5221,
5222, 5224, 5226, 5229, 5230, 5231, 5232, 5234, 5237, 5242, 5244, 5245, 5246,
5247, 5249,
5250, 5252, 5254, 5255, 5256, 5261, 5262, 5264, 5265, 5273, 5274, 5276, 5278,
5279, 5281,
5284, 5285, 5286, 5287, 5288, 5289, 5292, 5296, 5300, 5303, 5304, 5307, 5308,
5309, 5313,
5315, 5317, 5318, 5322, 5323, 5326, 5329, 5330, 5333, 5336, 5337, 5341, 5343,
5348, 5349,
5352, 5354, 5355, 5358, 5359, 5361, 5362, 5363, 5364, 5365, 5367, 5369, 5370,
5372, 5375,
5377, 5379, 5382, 5385, 5387, 5393, 5394, 5395, 5398, 5400, 5404, 5407, 5408,
5409, 5412,
5413, 5414, 5416, 5418, 5421, 5424, 5426, 5428, 5431, 5432, 5433, 5434, 5435,
5437, 5440,
CA 02528853 2005-12-23
5441, 5443, 5448, 5450, 5451, 5454, 5455, 5458, 5460, 5461, 5464, 5468, 5470,
5471, 5474,
5475, 5477, 5482, 5484, 5485, 5487, 5490, 5491, 5492, 5494, 5496, 5498, 5499,
5500, 5501,
5502, 5503, 5504, 5506, 5509, 5510, 5512, 5516, 5517, 5518, 5524, 5525, 5526,
5528, 5531,
5533, 5534, 5535, 5536, 5537, 5540, 5542, 5544, 5545, 5546, 5549, 5550, 5552,
5553, 5559,
5561, 5566, 5569, 5572, 5573, 5575, 5577, 5578, 5579, 5580, 5581, 5582, 5588,
5593, 5595,
5596, 5598, 5600, 5601, 5603, 5604, 5605, 5608, 5614, 5615, 5616, 5619, 5622,
5624, 5625,
5628, 5629, 5633, 5634, 5637, 5638, 5639, 5643, 5644, 5645, 5647, 5648, 5649,
5650, 5654,
5662, 5664, 5067, 5070, 5072, 5074, 5075, 5076, 5078, 5081, 5084, 5086, 5088,
5090, 5091,
5094, 5097, 5101, 5105, 5107, 5109, 5110, 5112, 5114, 5115, 5117, 5121, 5123,
5124, 5126,
5129, 5131, 5136, 5143, 5145, 5147, 5151, 5153, 5156, 5159, 5161, 5162, 5163,
5164, 5165,
5167, 5169, 5172, 5174, 5175, 5180, 5181, 5182, 5183, 5185, 5186, 5189, 5191,
5194, 5196,
5199, 5200, 5202, 5203, 5205, 5207, 5209, 5210, 5212, 5214, 5218, 5219, 5223,
5225, 5227,
5233, 5235, 5238, 5239, 5240, 5241, 5248, 5251, 5253, 5257, 5259, 5260, 5263,
5266, 5268,
5269, 5270, 5271, 5272, 5280, 5282, 5290, 5291, 5293, 5295, 5297, 5299, 5301,
5306, 5310,
5311, 5312, 5314, 5316, 5319, 5321, 5324, 5327, 5331, 5332, 5334, 5338, 5339,
5340, 5342,
5344, 5346, 5347, 5351, 5353, 5356, 5360, 5366, 5368, 5371, 5373, 5376, 5378,
5380, 5383,
5388, 5389, 5391, 5392, 5396, 5399, 5401, 5403, 5405, 5410, 5415, 5417, 5419,
5420, 5422,
5425, 5427, 5429, 5436, 5438, 5442, 5444, 5445, 5446, 5447, 5449, 5452, 5456,
5457, 5459,
5462, 5465, 5466, 5467, 5469, 5472, 5478, 5480, 5481, 5486, 5488, 5493, 5495,
5497, 5505,
5507, 5508, 5511, 5513, 5515, 5519, 5521, 5522, 5527, 5529, 5532, 5538, 5539,
5541, 5543,
5548, 5551, 5554, 5555, 5556, 5557, 5558, 5560, 5562, 5563, 5564, 5565, 5567,
5568, 5570,
5571, 5574, 5576, 5583, 5585, 5586, 5587, 5589, 5591, 5592, 5594, 5597, 5599,
5602, 5606,
5609, 5611, 5612, 5618, 5620, 5623, 5626, 5627, 5630, 5632, 5635, 5640, 5642,
5646, 5651,
5652, 5656, 5657, 5659, 5660, 5661, 5663, 5665, 5667, 5670, 5672, 5673, 5676,
5678, 5679,
'.5 5681, 5682, 5683, 5686, 5687, 5688, 5690, 5694, 5697, 5698, 5699, 5700,
5701, 5704, 5708,
5709, 5711, 5713, 5714, 5717, 5721, 5723, 5724, 5727, 5728, 5733, 5734, 5735,
5736, 5739,
5740, 5741, 5742, 5743, 5668, 5671, 5674, 5677, 5680, 5684, 5689, 5691, 5693,
5696, 5702,
5705, 5707, 5710, 5712, 5715, 5718, 5720, 5722, 5725, 5729, 5730, 5731, 5732,
5737, 5744,
5747, 5750, 5753, 5759, 5762, 5763, 5770, 5775, 5777, 5778, 5780, 5782, 5785,
5787, 5791,
86
CA 02528853 2005-12-23
5792, 5793, 5794, 5797, 5801, 5803, 5808, 5810, 5812, 5814, 5816, 5817, 5824,
5832, 5833,
5834, 5835, 5838, 5840, 5841, 5844, 5849, 5853, 5854, 5857, 5859, 5860, 5862,
5863, 5865,
5868, 5874, 5882, 5883, 5885, 5887, 5894, 5895, 5896, 5897, 5902, 5904, 5906,
5909, 5912,
5915, 5918, 5919, 5920, 5922, 5923, 5926, 5927, 5928, 5930, 5932, 5933, 5934,
5935, 5939,
5940, 5946, 5947, 5948, 5949, 5952, 5954, 5955, 5956, 5958, 5959, 5961, 5962,
5964, 5965,
5968, 5970, 5971, 5972, 5973, 5977, 5978, 5980, 5983, 5986, 5987, 5992, 5993,
5996, 5997,
6000, 6002, 6003, 6004, 6005, 6006, 6008, 6009, 6010, 6011, 6012, 6013, 6017,
6018, 6019,
6020, 6025, 6027, 6030, 6033, 6035, 6036, 6039, 6041, 6042, 6045, 6049, 6050,
6051, 6055,
6059, 6063, 6064, 6065, 6067, 6070, 6072, 6073, 6075, 6077, 6078, 6081, 5745,
5748, 5751,
5754, 5756, 5758, 5760, 5764, 5766, 5767, 5768, 5769, 5771, 5773, 5774, 5776,
5779, 5781,
5783, 5786, 5788, 5789, 5790, 5795, 5796, 5798, 5800, 5802, 5804, 5806, 5807,
5809, 5811,
5813, 5818, 5820, 5821, 5822, 5823, 5825, 5826, 5827, 5828, 5830, 5831, 5836,
5837, 5839,
5842, 5843, 5845, 5846, 5847, 5848, 5850, 5852, 5855, 5856, 5858, 5861, 5864,
5866, 5867,
5870, 5871, 5872, 5873, 5875, 5876, 5877, 5878, 5879, 5880, 5881, 5884, 5886,
5888, 5889,
5890, 5891, 5892, 5893, 5898, 5899, 5900, 5903, 5905, 5907, 5910, 5913, 5916,
5921, 5924,
5929, 5931, 5936, 5937, 5938, 5942, 5943, 5944, 5945, 5950, 5953, 5957, 5960,
5963, 5966,
5969, 5975, 5976, 5981, 5984, 5988, 5990, 5991, 5994, 5998, 5999, 6001, 6007,
6014, 6015,
6016, 6021, 6023, 6024, 6026, 6028, 6031, 6032, 6034, 6037, 6038, 6040, 6043,
6046, 6048,
6053, 6054, 6056, 6058, 6060, 6062, 6066, 6068, 6074, 6076, 6079, 6083, 6084,
6085, 6089,
6090, 6093, 6096, 6098, 6100, 6106, 6110, 6111, 6114, 6118, 6121, 6127, 6130,
6133, 6135,
6137, 6138, 6141, 6143, 6145, 6148, 6150, 6151, 6152, 6153, 6154, 6156, 6159,
6160, 6162,
6163, 6166, 6167, 6168, 6169, 6170, 6171, 6172, 6175, 6177, 6180, 6183, 6187,
6188, 6190,
6193, 6194, 6195, 6196, 6198, 6201, 6202, 6204, 6207, 6211, 6212, 6214, 6215,
6216, 6218,
6219, 6220, 6222, 6225, 6226, 6230, 6235, 6239, 6241, 6242, 6243, 6244, 6245,
6247, 6250,
5 6252, 6254, 6259, 6260, 6261, 6264, 6265, 6268, 6270, 6273, 6275, 6278,
6280, 6282, 6286,
6287, 6288, 6291, 6293, 6298, 6299, 6300, 6302, 6303, 6305, 6307, 6308, 6312,
6313, 6314,
6315, 6316, 6317, 6318, 6320, 6321, 6323, 6325, 6327, 6328, 6334, 6335, 6337,
6340, 6341,
6344, 6348, 6350, 6353, 6354, 6357, 6360, 6362, 6364, 6368, 6372, 6374, 6377,
6378, 6383,
6386, 6392, 6394, 6398, 6400, 6404, 6405, 6409, 6410, 6411, 6417, 6418, 6424,
6426, 6429,
87
CA 02528853 2005-12-23
6431, 6432, 6433, 6435, 6436, 6437, 6438, 6440, 6442, 6445, 6446, 6448, 6449,
6452, 6455,
6457, 6458, 6461, 6462, 6463, 6464, 6465, 6470, 6473, 6475, 6478, 6480, 6481,
6484, 6485,
6486, 6489, 6491, 6492, 6494, 6498, 6502, 6504, 6505, 6507, 6511, 6513, 6514,
6086, 6088,
6091, 6094, 6097, 6099, 6101, 6103, 6105, 6108, 6112, 6115, 6116, 6117, 6119,
6120, 6122,
6123, 6125, 6126, 6128, 6131, 6132, 6134, 6136, 6139, 6142, 6144, 6146, 6149,
6155, 6157,
6161, 6164, 6173, 6174, 6176, 6178, 6181, 6182, 6184, 6186, 6189, 6191, 6192,
6197, 6199,
6200, 6205, 6206, 6208, 6210, 6213, 6221, 6223, 6224, 6227, 6229, 6231, 6232,
6233, 6234,
6236, 6237, 6238, 6240, 6246, 6248, 6251, 6253, 6255, 6256, 6257, 6258, 6262,
6266, 6269,
6271, 6272, 6274, 6276, 6277, 6281, 6283, 6284, 6285, 6289, 6292, 6294, 6295,
6296, 6297,
6301, 6304, 6306, 6309, 6310, 6311, 6319, 6322, 6324, 6326, 6329, 6330, 6331,
6333, 6336,
6338, 6342, 6345, 6347, 6349, 6351, 6355, 6356, 6358, 6359, 6361, 6365, 6367,
6369, 6371,
6373, 6375, 6376, 6379, 6381, 6382, 6384, 6387, 6388, 6389, 6390, 6393, 6395,
6397, 6399,
6401, 6402, 6403, 6406, 6407, 6412, 6413, 6414, 6415, 6416, 6419, 6420, 6421,
6422, 6423,
6425, 6427, 6428, 6434, 6441, 6443, 6444, 6447, 6450, 6451, 6453, 6459, 6460,
6466, 6467,
6468, 6469, 6471, 6472, 6474, 6476, 6479, 6482, 6487, 6490, 6493, 6496, 6497,
6499, 6500,
6501, 6503, 6506, 6508, 6510, 6512, 6515, 6518, 6521, 6522, 6523, 6524, 6528,
6532, 6535, -
6539, 6549, 6560, 6561, 6564, 6566, 6567, 6570, 6573, 6576, 6577, 6578, 6579,
6583, 6584,
6585, 6586, 6587, 6590, 6591, 6594, 6596, 6S97, 6603, 6605, 6608, 6609, 6610,
6611, 6612,
6615, 6625, 6630, 6632, 6633, 6634, 6638, 6639, 6642, 6643, 6644, 6647, 6650,
6655, 6657,
6658, 6659, 6661, 6663, 6664, 6665, 6669, 6671, 6674, 6677, 6678, 6679, 6680,
6681, 6682,
6686, 6687, 6688, 6689, 6690, 6691, 6693, 6695, 6696, 6697, 6699, 6701, 6703,
6704, 6705,
6706, 6707, 6708, 6710, 6711, 6712, 6713, 6717, 6718, 6720, 6721, 6722, 6725,
6727, 6732,
6734, 6735, 6739, 6741, 6745, 6748, 6751, 6755, 6759, 6762, 6764, 6767, 6770,
6772, 6773,
6774, 6777, 6778, 6782, 6785, 6786, 6787, 6790, 6791, 6794, 6795, 6796, 6798,
6800, 6802,
6803, 6806, 6811, 6814, 6815, 6818, 6824, 6829, 6834, 6835, 6842, 6516, 6519,
6525, 6526,
6527, 6529, 6531, 6533, 6536, 6537, 6538, 6540, 6541, 6542, 6543, 6544, 6545,
6546, 6547,
6548, 6550, 6551, 6552, 6553, 6554, 6556, 6558, 6562, 6563, 6565, 6568, 6571,
6572, 6575,
6580, 6582, 6588, 6589, 6592, 6595, 6598, 6599, 6600, 6601, 6602, 6604, 6606,
6607, 6613,
6614, 6617, 6618, 6619, 6620, 6621, 6623, 6624, 6627, 6628, 6629, 6631, 6635,
6640, 6641,
88
CA 02528853 2005-12-23
}
6645, 6646, 6649, 6651, 6652, 6653, 6656, 6660, 6662, 6666, 6668, 6670, 6672,
6675, 6676,
6683, 6685, 6692, 6694, 6698, 6700, 6702, 6709, 6714, 6715, 6716, 6719, 6723,
6726, 6728,
6730, 6731, 6736, 6738, 6740, 6742, 6744, 6746, 6747, 6749, 6752, 6754, 6756,
6757, 6758,
6760, 6761, 6765, 6769, 6775, 6779, 6781, 6783, 6784, 6789, 6792, 6797, 6799,
6801, 6804,
6805, 6807, 6809, 6810, 6812, 6816, 6817, 6819, 6821, 6822, 6825, 6827, 6828,
6830, 6831,
6832, 6833, 6836, 6837, 6838, 6839, 6840, 6841, 6843,
Detection. 6517, 6520, 6 530, 6534, 6555, 6557, 6559, 6569, 6574, 6581, 6593,
6616,
6622, 6626, 6636, 6637, 6648, 6654, 6667, 6673, 6684, 6724, 6729, 6733, 6737,
6743, 6750,
6753, 6763, 6766, 6768, 6771, 6776, 6780, 6788, 6793, 6808, 6813, 6820, 6823,
6826, 6844,
6848, 6850, 6852, 6854, 6857, 6859, 6861, 6862, 6865, 6868, 6870, 6871, 6872,
6874, 6875,
6876, 6880, 6883, 6886, 6887, 6888, 6890, 6891, 6894, 6896, 6897, 6898, 6899,
6900, 6901,
6902, 6904, 6905, 6906, 6910, 6912, 6917, 6920, 6845, 6847, 6849, 6851, 6853,
6855, 6858,
6860, 6863, 6866, 6869, 6873, 6877, 6879, 6881, 6882, 6884, 6889, 6892, 6893,
6895, 6907,
6908, 6909, 6911, 6913, 6915, 6916, 6918, 6921, 6924, 6927, 6928, 6933, 6935,
6937, 6939,
6940, 6943, 6946, 6947, 6948, 6949, 6951, 6953, 6954, 6955, 6959, 6961, 6964,
6965, 6968,
6969, 6972, 6973, 6976, 6979, 6980, 6922, 6925, 6929, 6930, 6931, 6932, 6934,
6936, 6942,
6944, 6945, 6950, 6952, 6956, 6957, 6958, 6960, 6962, 6963, 6966, 6967, 6970,
6971, 6974,
6975, 6977, 6981, 6983, 6986, 6992, 6995, 6998, 7002, 7006, 7013, 7017, 7022,
7024, 7027, .
7030, 7032, 7036, 7037, 7040, 7050, 7052, 7055, 7056, 7058, 70S9, 7060, 7064,
7067, 7072,
7074, 7076, 7077, 7084, 7085, 7086, 7087, 7088, 7089, 7090, 7091, 7096, 7097,
7100, 7102,
7109, 7110, 7113, 7114, 7115, 7118, 7121, 7123, 7124, 7131, 7133, 7135, 7136,
7138, 7139,
7140, 7144, 7146, 7148, 7149, 7158, 7159, 7160, 7163, 7165, 7166, 7168, 7172,
7175, 7178,
7180, 7181, 7183, 7185, 7186, 7187, 7192, 7194, 7199, 7202, 7203, 7204, 7205,
7207, 7208,
7213, 7214, 7216, 7220, 7221, 7227, 7228, 7230, 7233, 7235, 7237, 7239, 7243,
7245, 7246,
7248, 7249, 7251, 7255, 7256, 7260, 7263, 7264, 7265, 7266, 7268, 7269, 7272,
7276, 7277,
7278, 7279, 7280, 7281, 7283, 7284, 7287, 7291, 7295, 7299, 7302, 7303, 7306,
7309, 7310,
7311, 7314, 7315, 7320, 7321, 7324, 7325, 7329, 7331, 7332, 7334, 7336, 7339,
7340, 7341,
7344, 7350, 7353, 7355, 7358, 7360, 7362, 7372, 7374, 7375, 7378, 7381, 7385,
7387, 7390,
7391, 7392, 7394, 7397, 7401, 7402, 7405, 7410, 7413, 7415, 7416, 7417, 7420,
6984, 6987,
89
CA 02528853 2005-12-23
6989, 6990, 6991, 6993, 6996, 6999, 7001, 7003, 7005, 7007, 7009, 7010, 7011,
7012, 7014,
7016, 7018, 7020, 7023, 7025, 7026, 7028, 7029, 7031, 7033, 7034, 7035, 7038,
7039, 7041,
7042, 7043, 7044, 7045, 7046, 7047, 7048, 7049, 7051, 7053, 7054, 7057, 7061,
7062, 7063,
7065, 7066, 7068, 7070, 7071, 7073, 7075, 7078, 7080, 7081, 7082, 7083, 7092,
7093, 7094,
7095, 7098, 7101, 7103, 7105, 7106, 7107, 7108, 7111, 7112, 7116, 7119, 7122,
7125, 7126,
7127, 7128, 7129, 7130, 7132, 7134, 7137, 7141, 7142, 7143, 7147, 7150, 7152,
7154, 7155,
7156, 7157, 7161, 7167, 7170, 7171, 7173, 7174, 7176, 7177, 7179, 7182, 7184,
7188, 7189,
7190, 7191, 7193, 7195, 7196, 7197, 7198, 7200, 7206, 7210, 7211, 7212, 7215,
7217, 7218,
7219, 7222, 7223, 7224, 7225, 7226, 7229, 7231, 7232, 7236, 7240, 7241, 7242,
7244, 7247, -
7250, 7253, 7254, 7257, 7258, 7259, 7261, 7262, 7267, 7270, 7271, 7273, 7274,
7275, 7282, '
7285, 7289, 7290, 7292, 7293, 7294, 7296, 7297, 7298, 7301, 7304, 7307, 7308,
7312, 7316,
7317, 7318, 7319, 7322, 7326, 7328, 7330, 7333, 7335, 7337, 7342, 7345, 7347,
7348, 7349,
7351, 7354, 7356, 7357, 7359, 7361, 7363, 7364, 7365, 7366, 7367, 7368, 7369,
7370, 7371,
7373, 7376, 7379, 7382, 7383, 7384, 7386, 7388, 7389, 7393, 7395, 7398, 7400,
7403, 7404,
7407, 7408, 7409, 7411, 7419, 7421, 7422, 7425, 7427, 7435, 7438, 7439, 7440,
7442, 7444,
7448, 7449, 7456, 7459, 7462, 7463, 7465, 7466, 7467, 7470, 7471, 7474, 7476,
7477, 7480,
7481, 7485, 7486, 7489, 7490, 7493, 7496, 7500, 7503, 7508, 7512, 7518, 7519,
7520, 7522,
7524, 7525, 7527, 7528, 7529, 7532, 7540, 7542, 7543, 7545, 7549, 7552, 7554,
7423, 7426,
7428, 7430, 7431, 7432, 7433, 7434, 7436, 7445, 7446, 7447, 7450, 7451, 7452,
7453, 7454,
7455, 7457, 7460, 7464, 7468, 7472, 7475, 7478, 7479, 7482, 7483, 7484, 7487,
7488, 7492,
7494, 7495, 7497, 7498, 7499, 7501, 7505, 7506, 7507, 7509, 7510, 7511, 7513,
7515, 7516,
7517, 7521, 7523, 7526, 7530, 7533, 7534, 7535, 7536, 7537, 7538, 7539, 7541,
7544, 7546,
7547, 7548, 7550, 7555, 7558, 7559, 7560, 7565, 7566, 7567, 7570, 7571, 7573,
7574, 7575,
7576, 7577, 7578, 7579, 7580, 7581, 7582, 7585, 7586, 7587, 7588, 7589, 7590,
7596, 7597,
7598, 7600, 7603, 7606, 7609, 7610, 7615, 7618, 7620, 7621, 7623, 7627, 7629,
7633, 7635,
7636, 7643, 7647, 7651, 7655, 7661, 7666, 7672, 7674, 7676, 7677, 7682, 7683,
7685, 7687,
7689, 7690, 7691, 7694, 7699, 7701, 7702, 7704, 7707, 7709, 7711, 7712, 7713,
7714, 7716,
7718, 7720, 7726, 7728, 7729, 7733, 7734, 7735, 7736, 7738, 7740, 7741, 7744,
7746, 7747,
7748, 7749, 7750, 7751, 7752, 7753, 7754, 7755, 7756, 7758, 7556, 7561, 7563,
7568, 7583,
CA 02528853 2005-12-23
7584, 7591, 7593, 7594, 7595, 7599, 7601, 7604, 7607, 7608, 7611, 7613, 7614,
7616, 7617,
7619, 7622, 7624, 7626, 7628, 7631, 7637, 7638, 7639, 7640, 7641, 7642, 7644,
7645, 7646,
7649, 7650, 7652, 7654, 7656, 7657, 7658, 7659, 7660, 7662, 7663, 7665, 7667,
7669, 7670,
7671, 7673, 7675, 7678, 7679, 7680, 7681, 7684, 7686, 7688, 7692, 7695, 7697,
7698, 7700,
7703, 7705, 7708, 7710, 7715, 7717, 7719, 7721, 7722, 7723, 7724, 7725, 7727,
7730, 7731,
7732, 7737, 7739, 7742, 7743, 7745, 7757, 7759, 7760, 7761, 7764, 7770, 7774,
7775, 7778,
7782, 7786, 7788, 7795, 7797, 7799, 7805, 7807, 7809, 7810, 7811, 7815, 7817,
7819, 7820,
7822, 7823, 7824, 7828, 7832, 7834, 7837, 7839, 7843, 7845, 7851, 7852, 7857,
7860, 7863,
7865, 7868, 7874, 7881, 7885, 7889, 7892, 7894, 7896, 7900, 7902, 7904, 7907,
7909, 7910,
7913, 7917, 7922, 7925, 7926, 7928, 7933, 7934, 7937, 7941, 7945, 7946, 7948,
7950, 7951,
7952, 7953, 7954, 7955, 7956, 7957, 7963, 7964, 7968, 7971, 7972, 7973, 7976,
7979, 7980,
7981, 7982, 7983, 7985, 7986, 7987, 7988, 7991, 7762, 7765, 7767, 7768, 7771,
7773, 7776,
7779, 7781, 7783, 7784, 7785, 7789, 7791, 7792, 7793, 7794, 7796, 7798, 7800,
7802, 7803,
7808, 7812, 7813, 7814, 7816, 7818, 7821, 7825, 7827, 7829, 7830, 7833, 7835,
7836, 7838,
7840, 7841, 7842, 7844, 7846, 7847, 7848, 7849, 7853, 7854, 7855, 7856, 7858,
7861, 7864,
7866, 7869, 7870, 7871, 7872, 7873, 7875, 7877, 7878, 7879, 7880, 7882, 7884,
7886, 7888,
7890, 7897, 7899, 7903, 7905, 7912, 7914, 7916, 7918, 7919, 7920, 7921, 7923,
7929, 7931,
7932, 7936, 7938, 7939, 7940, 7942, 7943, 7944, 7947, 7949, 7958, 7960, 7962,
7965, 7967,
7969, 7970, 7974, 7977, 7989, 7990, 7992, 7995, 7996, 8000, 8003, 8006, 8007,
8010, 8012,
8015, 8016, 8018, 8019, 8026, 8027, 8030, 8034, 8035; 8038, 8039, 8043, 8044,
8046, 8049,
8050, 8052, 8053, 8055, 8056, 8062, 8064, 8066, 8067, 8068, 8069, 8070, 8071,
8074, 8076,
8077, 8078, 8079, 8080, 8081, 7993, 7997, 7999, 8001, 8004, 8005, 8008, 8009,
8011, 8013,
8017, 8020, 8021, 8022, 8023, 8024, 8025, 8028, 8031, 8033, 8036, 8040, 8042,
8047, 8051,
8054, 8057, 8059, 8060, 8061, 8065, 8072, 8073, 8075, 8082, 8083, 8084, 8085,
8087, 8088,
8089, 8090, 8093, 8095, 8098, 8100, 8101, 8102, 8104, 8107, 8110, 8111, 8114,
8115, 8116,
8118, 8122, 8124, 8126, 8127, 8129, 8132, 8134, 8136, 8138, 8142, 8143, 8144,
8146, 8151,
8157, 8160, 8163, 8165, 8168, 8169, 8170, 8173, 8174, 8175, 8176, 8178, 8181,
8185, 8187,
8188, 8189, 8190, 8191, 8193, 8194, 8200, 8203, 8206, 8208, 8209, 8212, 8214,
8216, 8217,
8220, 8222, 8224, 8228, 8230, 8091, 8094, 8096, 8099, 8103, 8105, 8108, 8112,
8113, 8117,
91
CA 02528853 2005-12-23
8119, 8121, 8123, 8125, 8128, 8130, 8131, 8133, 8135, 8137, 8139, 8145, 8147,
8148, 8149,
8150, 8152, 8153, 8154, 8155, 8156, 8158, 8161, 8164, 8166, 8167, 8171, 8177,
8179, 8180,
8182, 8183, 8184, 8186, 8192, 8195, 8197, 8198, 8199, 8201, 8202, 8204, 8207,
8210, 8213,
8218, 8221, 8223, 8225, 8227, 8229, 8231, 8232, 8235, 8236, 8237, 8238, 8244,
8245, 8247,
8250, 8251, 8253, 8254, 8255, 8256, 8258, 8259, 8260, 8263, 8267, 8269, 8270,
8273, 8274,
8277, 8280, 8281, 8289, 8292, 8293, 8294, 8295, 8296, 8297, 8298, 8301, 8305,
8306, 8310,
8311, 8312, 8315, 8317, 8318, 8322, 8323, 8324, 8327, 8328, 8329, 8330, 8333,
8336, 8339,
8340, 8347, 8349, 8350, 8352, 8353, 8356, 8359, 8360, 8366, 8369, 8370, 8372,
8374, 8376,
8377, 8379, 8381, 8382, 8383, 8233, 8239, 8240, 8242, 8243, 8246, 8248, 8252,
8257, 8261,
8264, 8266, 8268, 8271, 8275, 8278, 8282, 8284, 8285, 8286, 8287, 8288, 8291,
8299, 8302,
8303, 8304, 8307, 8309, 8313, 8314, 8316, 8319, 8321, 8325, 8326, 8331, 8332,
8334, 8335,
8337, 8341, 8342, 8343, 8344, 8345, 8346, 8348, 8351, 8354, 8357, 8361, 8362,
8363, 8364,
8365, 8367, 8371, 8373, 8375, 8378, 8384, 8387, 8390, 8391, 8397, 8398, 8401,
8402, 8405,
8406, 8409, 8414, 8418, 8419, 8422, 8425, 8428, 8431, 8432, 8433, 8435, 8437,
8440, 8444,
8449, 8452, 8454, 8455, 8457, 8462, 8464, 8466, 8468, 8469, 8472, 8475, 8476,
8478, 8479,
8481, 8484, 8487, 8488, 8490, 8493, 8496, 8497, 8498, 8501, 8505, 8508, 8511,
8513, 8515,
8517, 8518, 8519, 8521, 8522, 8523, 8526, 8528, 8530, 8531, 8532, 8534, 8536,
8541, 8544,
8547, 8551, 8554, 8556, 8558, 8560, 8562, 8563, 8566, 8567, 8568, 8570, 8571,
8574, 8575,
8576, 8577, 8580, 8581, 8585, 8588, 8591, 8594, 8601, 8602, 8603, 8608, 8609,
8613, 8614,
8617, 8618, 8619, 8620, 8623, 8624, 8628, 8629, 8630, 8633, 8634, 8635, 8636,
8638, 8643,
8645, 8648, 8654, 8657, 8658, 8659, 8660, 8661, 8662, 8666, 8670, 8672, 8674,
8676, 8681,
8683, 8686, 8688, 8689, 8691, 8692, 8693, 8695, 8696, 8700, 8701, 8702, 8703,
8704, 8705,
8709, 8716, 8717, 8719, 8722, 8725, 8727, 8728, 8729, 8732, 8733, 8734, 8735,
8736, 8385,
8388, 8392, 8394, 8395, 8399, 8403, 8407, 8410, 8411, 8412, 8413, 8415, 8416,
8420, 8423,
8424, 8426, 8429, 8434, 8436, 8438, 8442, 8445, 8446, 8447, 8448, 8450, 8451,
8456, 8458,
8460, 8461, 8463, 8465, 8467, 8470, 8473, 8474, 8477, 8480, 8482, 8485, 8486,
8489, 8491,
8492, 8494, 8499, 8500, 8502, 8503, 8504, 8506, 8510, 8512, 8514, 8516, 8520,
8524, 8527,
8529, 8533, 8535, 8537, 8539, 8542, 8545, 8546, 8548, 8550, 8552, 8555, 8557,
8564, 8565,
8569, 8572, 8578, 8582, 8583, 8586, 8587, 8589, 8592, 8595, 8596, 8597, 8598,
8599, 8600,
92
CA 02528853 2005-12-23
8604, 8606, 8607, 8610, 8612, 8615, 8616, 8621, 8625, 8627, 8631, 8632, 8637,
8639, 8640,
8641, 8642, 8644, 8646, 8647, 8649, 8650, 8651, 8652, 8653, 8655, 8663, 8665,
8667, 8668,
8669, 8671, 8673, 8675, 8677, 8678, 8679, 8680, 8682, 8684, 8685, 8687, 8690,
8694, 8697,
8698, 8699, 8706, 8707, 8708, 8710, 8711, 8712, 8713, 8714, 8715, 8718, 8720,
8723, 8726,
8730, 8737, 8738, 8741, 8744, 8747, 8750, 8751, 8753, 8754, 8755, 8756, 8759,
8760, 8762,
8763, 8766, 8767, 8768, 8769, 8770, 8772, 8775, 8776, 8777, 8779, 8782, 8783,
8784, 8785,
8786, 8787, 8788, 8789, 8790, 8793, 8794, 8796, 8798, 8799, 8801, 8803, 8804,
8806, 8807,
8809, 8812, 8813, 8815, 8820, 8823, 8826, 8829, 8830, 8832, 8835, 8836, 8837,
8842, 8845,
8846, 8847, 8851, 8853, 8856, 8857, 8859, 8861, 8866, 8739, 8878, 8879, 8880,
8884, 8891,
8893, 8895, 8898, 8900, 8901, 8909, 8912, 8739, 8742, 8745, 8748, 8749, 8752,
8757, 8758,
8761, 8764, 8771, 8773, 8778, 8780, 8791, 8797, 8800, 8802, 8805, 8808, 8810,
8814, 8816,
8818, 8819, 8821, 8824, 8827, 8828, 8831, 8833, 8838, 8840, 8841, 8755, 8843,
8844, 8848,
8850, 8852, 8854, 8855, 8863, 8864, 8865, 8867, 8868, 8869, 8870, 8871, 8872,
8873, 8874,
8876, 8877, 8881, 8882, 8883, 8885, 8887, 8888, 8889, 8890, 8892, 8894, 8896,
8897, 8899,
8902, 8785, 8903, 8904, 8905, 8906, 8907, 8908, 8911, 8913, 8917, 8920, 8923,
8924, 8929,
8931, 8934, 8935, 8936, 8914, 8916, 8918, 8921, 8925, 8927, 8928, 8930, 8932,
8933, 8937,
8980, 8984, 8988, 8989, 8998, 9001, 9002, 9005, 9007, 9008, 9012, 9013, 9016,
9019, 9020,
9022, 9023, 9025, 9028, 9029, 9032, 9033, 9034, 9035, 9037, 9038, 9044, 9046,
9047, 9048,
9049, 9054, 8981, 8983, 8985, 8987, 8990, 8992, 8993, 8994, 8995, 8997, 8999,
9003, 9004,
9006, 9009, 9010, 9011, 9014, 9017, 9018, 9021, 9024, 9026, 9030, 9036, 9039,
9041, 9042,
9043, 9045, 9050, 9051, 9052, 9053, 9055, 9058, 9060, 9063, 9066, 9067, 9068,
9069, 9070,
9071, 9074, 9075, 9077, 9078, 9079, 9080, 9082, 9084, 9088, 9089, 9090, 9099,
9103, 9104,
9105, 9107, 9111, 9056, 9061, 9064, 9072, 9081, 9083, 9085, 9086, 9087, 9091,
9092, 9093,
9094, 9095, 9096, 9097, 9098, 9100, 9102, 9106, 9108, 9109, 9110, 9112, 9113,
9116, 9118,
9125, 9126, 9128, 9129, 9130, 9136, 9141, 9143, 9148, 9150, 9153, 9157, 9158,
9159, 9163,
9164, 9165, 9167, 9168, 9171, 9173, 9177, 9180, 9181, 9182, 9183, 9185, 9187,
9188, 9189,
9191, 9192, 9193, 9194, 9197, 9198, 9200, 9206, 9207, 9208, 9209, 9211, 9216,
9218, 9219,
9220, 9221, 9222, 9223, 9226, 9228, 9229, 9230, 9231, 9232, 9233, 9234, 9237,
9238, 9240,
9243, 9246, 9248, 9253, 9256, 9257, 9260, 9263, 9264, 9266, 9269, 9271, 9279,
9280, 9281,
93
CA 02528853 2005-12-23
9282, 9283, 9284, 9290, 9114, 9119, 9121, 9122, 9123, 9124, 9127, 9131, 9133,
9134, 9135,
9137, 9138, 9139, 9140, 9142, 9144, 9146, 9147, 9149, 9151, 9152, 9154, 9155,
9156, 9160,
9162, 9166, 9169, 9172, 9174, 9175, 9176, 9178, 9184, 9186, 9190, 9195, 9199,
9201, 9202,
9203, 9204, 9205, 9212, 9214, 9215, 9217, 9224, 9227, 9235, 9239, 9241, 9244,
9247, 9249,
9251, 9252, 9254, 9258, 9261, 9265, 9267, 9270, 9273, 9274, 9275, 9276, 9277,
9278, 9285,
9286, 9287, 9288, 9289, 9291, 9292, 9295, 9297, 9300, 9302, 9303, 9304, 9307,
9308, 9310,
9311, 9313, 9319, 9321, 9323, 9324, 9328, 9333, 9334, 9337, 9338, 9339, 9342,
9344, 9348,
9351, 9352, 9354, 9355, 9357, 9360, 9367, 9371, 9374, 9377, 9379, 9383, 9384,
9385, 9388,
9389, 9390, 9393, 9394, 9398, 9401, 9402, 9406, 9408, 9409, 9410, 9413, 9415,
9416, 9417,
9418, 9419, 9421, 9422, 9425, 9429, 9433, 9434, 9435, 9436, 9437, 9439, 9440,
9446, 9452,
9453, 9454, 9455, 9457, 9458, 9459, 9462, 9466, 9467, 9468, 9470, 9474, 9479,
9482, 9487,
9488, 9489, 9492, 9495, 9497, 9499, 9500, 9504, 9505, 9508, 9513, 9514, 9519,
9522, 9523,
9524, 9526, 9528, 9532, 9533, 9534, 9535, 9537, 9538, 9541, 9542, 9543, 9545,
9546, 9548,
9293, 9296, 9298, 9301, 9305, 9309, 9312, 9314, 9316, 9317, 9318, 9320, 9322,
9325, 9326,
9327, 9329, 9331, 9332, 9335, 9336, 9340, 9345, 9347, 9349, 9353, 9356, 9358,
9361, 9363,
9364, 9365, 9368, 9370, 9372, 9375, 9376, 9378, 9380, 9382, 9386, 9391, 9395,
9397, 9399,
9400, 9403, 9405, 9407, 9411, 9412, 9414, 9420, 9423, 9424, 9426, 9428, 9430,
9431, 9432,
9438, 9441, 9443, 9444, 9445, 9447, 9448, 9449, 9450, 9451, 9456, 9460, 9461,
9464, 9465,
9469, 9471, 9473, 9475, 9477, 9478, 9480, 9483, 9485, 9490, 9493, 9494, 9496,
9498, 9501,
9502, 9503, 9506, 9509, 9511, 9512, 9515, 9516, 9517, 9518, 9520, 9521, 9525,
9527, 9529,
9530, 9536, 9539, 9544, 9547, 9549, 9550, 9551, 9555, 9556, 9557, 9559, 9560,
9563, 9564,
9565, 9566, 9569, 9570, 9573, 9577, 9578, 9579, 9580, 9585, 9586, 9587, 9588,
9591, 9593,
9596, 9552, 9554, 9558, 9561, 9567, 9571, 9574, 9576, 9581, 9583, 9584, 9589,
9592, 9594,
9595, 9598, 9602, 9604, 9605, 9608, 9612, 9613, 9614, 9617, 9618, 9621, 9622,
9625, 9626,
9627, 9632, 9633, 9638, 9640, 9643, 9644, 9645, 9646, 9651, 9654, 9656, 9657,
9658, 9661,
9662, 9665, 9666, 9667, 9668, 9672, 9673, 9675, 9676, 9677, 9680, 9683, 9686,
9688, 9691,
9694, 9698, 9699, 9702, 9705, 9707, 9710, 9711, 9715, 9719, 9720, 9722, 9723,
9726, 9729,
9732, 9733, 9735, 9737, 9738, 9741, 9744, 9745, 9746, 9747, 9748, 9749, 9750,
9754, 9755,
9756, 9758, 9759, 9768, 9771, 9772, 9776, 9777, 9781, 9782, 9784, 9787, 9789,
9790, 9792,
94
CA 02528853 2005-12-23
9796, 9797, 9800, 9805, 9806, 9812, 9815, 9818, 9821, 9823, 9825, 9827, 9831,
9832, 9835,
9837, 9839, 9841, 9842, 9844, 9845, 9851, 9853, 9854, 9855, 9857, 9860, 9862,
9863, 9865,
9871, 9873, 9875, 9878, 9879, 9599, 9601, 9603, 9606, 9607, 9609, 9611, 9615,
9619, 9623,
9624, 9628, 9630, 9631, 9634, 9635, 9637, 9639, 9641, 9647, 9649; 9650, 9652,
9653, 9659,
9660, 9663, 9664, 9670, 9671, 9679, 9682, 9684, 9687; 9690, 9692, 9693, 9695,
9697, 9700,
9701, 9703, 9706, 9708, 9712, 9713, 9714, 9716, 9717, 9718, 9721, 9724, 9727,
9730, 9734,
9739, 9742, 9751, 9753, 9757, 9760, 9761, 9762, 9763, 9764, 9765, 9766, 9767,
9769, 9773,
9775, 9778, 9780, 9783, 9786, 9788, 9791, 9793, 9794, 9795, 9798, 9801, 9803,
9804, 9807,
9808, 9809, 9810, 9811, 9813, 9816, 9817, 9819, 9820, 9822, 9826, 9828, 9830;
9833, 9836,
9838, 9840, 9843, 9846, 9847, 9848, 9849, 9850, 9852, 9856, 9858, 9859, 9861,
9864, 9866,
9867, 9868, 9869, 9870, 9872, 9876, 9877, 9880, 9881, 9882, 9886, 9890, 9891,
9894, 9900,
9902, 9904, 9908, 9911, 9912, 9915, 9917, 9919, 9923, 9925, 9928, 9935, 9936,
9940, 9943,
9949, 9953, 9954, 9956, 9957, 9958, 9962, 9963, 9964, 9968, 9969, 9975, 9978,
9981, 9988,
9989, 9990, 9991, 9992, 9995, 9997, 10011, 10012, 10014, 10017, 10018, 10031,
10032,
10033, 10034, 10035, 10037, 10041, 10042, 10046, 10050, 10054, 10058, 10059,
9883, 9885,
9887, 9889, 9892, 9893, 9895, 9896, 9897, 9898, 9899, 9901, 9903, 9905, 9907,
9909, 9913,
9914, 9916, 9918, 9920, 9921, 9922, 9924, 9926, 9929, 9931, 9932, 9933, 9934,
9937, 9938,
9939, 9941, 9942, 9944, 9945, 9946, 9947, 9948, 9950, 9952, 9955, 9959, 9960,
9961, 9966,
9967, 9970, 9972, 9973, 9974, 9976, 9979, 9980, 9982, 9983, 9984, 9985, 9986,
9987, 9993,
9994, 9996, 9998, 9999, 10000, 10001, 10002, 10003, 10004, 10005, 10006,
10007, 10008,
10009, 10010, 10013, 10015, 10016, 10019, 10020, 10021, 10022, 10023, 10025,
10026,
10027, 10028, 10030, 10036, 10038, 10040, 10043, 10044, 10045, 10047, 10048,
10049,
10051, 10053, 10055, 10056, 10057, 10060, 10061, 10064, 10067, 10068, 10070,
10071,
10072, 10074, 10079, 10081, 10083, 10088, 10091, 10095, 10096, 10097, 10101,
10103,
10105, 10106, 10109, 10110, 10111, 10113, 10116, 10118, 10120, 10122, 10125,
10126,
10127, 10131, 10134, 10137, 10138, 10144, 10147, 10149, 10151, 10152, 10155,
10156, '
10159, 10160, 10169, 10172, 10174, 10175, 10178, 10181, 10186, 10188, 10189,
10190,
10191, 10192, 10196, 10199, 10203, 10204, 10210, 10211, 10216, 10218, 10221,
10224,
10225, 10226, 10229, 10230, 10231, 10232, 10234, 10239, 10241, 10244, 10252,
10253,
CA 02528853 2005-12-23
10257, 10258, 10259, 10261, 10266, 10272, 10276, 10062, 10065, 10069, 10075,
10077,
10078, 10080, 10082, 10084, 10086, 10087, 10089, 10090, 10092, 10093, 10094,
10098,
10099, 10100, 10102, 10104, 10107, 10112, 10114, 10117, 10119, 10121, 10123,
10124,
10129, 10130, 10132, 10133, 10135, 10139, 10140, 10141, 10142, 10143, 10145,
10148,
10150, 10153, 10154, 10157, 10161, 10163, 10164, 10165, 10166, 10167, 10170,
10173,
10176, 10177, 10179, 10182, 10183, 10184, 10187, 10193, 10194, 10195, 10197,
10198,
10200, 10201, 10202, 10205, 10206, 10207, 10209, 10212, 10213, 10214, 10215,
10219,
10220, 10222, 10223, 10227, 10228, 10233, 10235, 10236, 10237, 10240, 10242,
10243,
10245, 10247, 10248, 10249, 10251, 10254, 10255, 10256, 10260, 10262, 10263,
10264,
10265, 10267, 10268, 10269, 10270, 10271, 10273, 10274, 10275, 10277, 10278,
10279,
10282, 10285, 10287, 10291, 10292, 10297, 10299, 10301, 10304, 10306, 10308,
10310,
10314, 10317, 10318, 10320, 10322, 10327, 10329, 10330, 10333, 10335, 10336,
10337,
10340, 10342, 10348, 10350, 10351, 10354, 10358, 10359, 10360, 10362, 10366,
10369,
10372, 10373, 10374, 10376, 10378, 10383, 10385, 10391, 10398, 10403, 10407,
10410,
10413, 10415, 10419, 10422, 10425, 10427, 10428, 10429, 10430, 10432, 10433,
10434,.
10437, 10438, 10439, 10442, 10443, 10445, 10446, 10447, 10448, 10452, 10453,
10454,
10455, 10456, 10460, 10463, 10466, 10467, 10469, 10470, 10473, 10475, 10477,
10480,
10484, 10488, 10490, 10494, 10495, 10497, 10501, 10502, 10503, 10507, 10508,
10510,
10517, 10518, 10520, 10522, 10524, 10525, 10528, 10529, 10533, 10536, 10537,
10540,
10541, 10545, 10547, 10551, 10553, 10556, 10559, 10560, 10563, 10564, 10565,
10568,
10571, 10574, 10576, 10579, 10581, 10583, 10588, 10589, 10590, 10592, 10593,
10596,
10597, 10601, 10602, 10603, 10605, 10607, 10609, 10610, 10612, 10613, 10617,
10618,
10621, 10624, 10627, 10630, 10632, 10634, 10637, 10638, 10639, 10640, 10641,
10643,
10645, 10647, 10653, 10655, 10657, 10659, 10661, 10662, 10663, 10664, 10665,
10666,
10667, 10668, 10669, 10672, 10673, 10674, 10675, 10280, 10283, 10286, 10288,
10290,
10293, 10295, 10298, 10300, 10302, 10305, 10307, 10309, 10311, 10312, 10313,
10315,
10319, 10321, 10323, 10325, 10326, 10328, 10331, 10338, 10341, 10343, 10344,
10345,
10346, 10347, 10349, 10352, 10355, 10356, 10357, 10361, 10363, 10365, 10367,
10370,
10375, 10377, 10379, 10380, 10381, 10382, 10384, 10386, 10387, 10388, 10390,
10392,
96
-
CA 02528853 2005-12-23
10394, 10395, 10399, 10401, 10402, 10404, 10406, 10408, 10411, 10412, 10414,
10416,
10417, 10418, 10420, 10423, 10426, 10431, 10435, 10440, 10444, 10449, 10451,
10457,
10459, 10461, 10462, 10464, 10465, 10468, 10471, 10476, 10478, 10481, 10483,
10485,
10487, 10489, 10491, 10492, 10493, 10498, 10499, 10500, 10504, 10506, 10509,
10511,
10513, 10514, 10515, 10516, 10519, 10521, 10523, 10526, 10530, 10531, 10532,
10534,
10535, 10538, 10539, 10542, 10543, 10544, 10546, 10548, 10550, 10552, 10554,
10557,
10558, 10561, 10566, 10569, 10572, 10573, 10577, 10578, 10580, 10582, 10584,
10586,
10594, 10598, 10600, 10604, 10606, 10611, 10614, 10616, 10619, 10622, 10625,
10626,
10628, 10631, 10633, 10635, 10642, 10644, 10646, 10648, 10649, 10650, 10651,
10652,
10654, 10656, 10658, 10660, 10670, 10671, 10676, 10678, 10684, 10679, 10681,
10682,
10683, 10685, 10687, 10688, 10689, 10690, 10691, 10693, 10696, 10699, 10703,
10704,
10709, 10711, 10712, 10713, 10720, 10721, 10722, 10724, 10725, 10726, 10728,
10729,
10734, 10740, 10742, 10743, 10744, 10745, 10746, 10748, 10749, 10751, 10754,
10758,
10759, 10760, 10764, 10768, 10769, 10772, 10775, 10780, 10781, 10783, 10786,
10791,
10796, 10801, 10808; 10817, 10818, 10819, 10821, 10823, 10826, 10827, 10829,
10830,
10834, 10843, 10844, 10847, 10848, 10849, 10853, 10856, 10858, 10866, 10869,
10872;
10874, 10877, 10878, 10879, 10882, 10884, 10885, 10889, 10892, 10893, 10896,
10900;
10901, 10903, 10905, 10907, 10909, 10910, 10911, 10914, 10916, 10919, 10926;
10930,
10932, 10933, 10934, 10938, 10941, 10942, 10943, 10944, 10946, 10947, 10953,
10955,
10956, 10958, 10959, 10962, 10967, 10968, 10972, 10975, 10977, 10978, 10981,
10984,
10985, 10988, 10992, 10995, 10997, 10999, 11000, 11001, 11006, 11009, 11013,
11016,
11020, 11025, 11033, 11036, 11040, 11041, 11042, 11043, 11045, 11046, 11048,
11052,
11055, 11060, 11061, 11062, 11064, 11065, 11067, 11070, 11071, 11072, 11078,
11079,
11081, 11085, 11093, 10694, 10697, 10700, 10702, 10705, 10707, 10710, 10714,
10716,
10717, 10718, 10719, 10723, 10727, 10730, 10732, 10733, 10735, 10737, 10738,
10739,
10741, 10747, 10750, 10752, 10755, 10757, 10761, 10762, 10763, 10765, 10767,
10770,
10773, 10776, 10778, 10779, 10782, 10784, 10788, 10789, 10790, 10792, 10793,
10794,
10798, 10799, 10800, 10802, 10804, 10805, 10806, 10807, 10809, 10811, 10812,
10813,
10814, 10815, 10816, 10820, 10822, 10824, 10831, 10833, 10835, 10837, 10838,
10839,
97
CA 02528853 2005-12-23
10840, 10841, 10842, 10845, 10846, 10850, 10851, 10854, 10857, 10859, 10860,
10861,
10862, 10863, 10864, 10865, 10868, 10870, 10871, 10873, 10875, 10880, 10881,
10883,
10886, 10888, 10890, 10894, 10897, 10899, 10902, 10904, 10906, 10908, 10912,
10915,
10917, 10920, 10922, 10924, 10925, 10927, 10928, 10929, 10931, 10935, 10937,
10939,
10945, 10948, 10950, 10951, 10952, 10954, 10957, 10960, 10963, 10965, 10966,
10969,
10970, 10971, 10973, 10976, 10979, 10980, 10982, 10986, 10987, 10989, 10991,
10993,
10996, 10998, 11002, 11004, 11005, 11007, 11010, 11012, 11014, 11015, 11017,
11019,
11021, 11022, 11023, 11026, 11027, 11028, 11029, 11030, 11031, 11032, 11034,
11037,
11039, 11044, 11047, 11049, 11050, 11051, 11053, 11054, 11056, 11058, 11059,
11063,
11066, 11068, 11069, 11073, 11074, 11075, 11076, 11077, 11080, 11082, 11083,
11084,
11086, 11087, 11088, 11089, 11090, 11091, 11092, 11094, 11376, 11381, 11382,
11383,
11384, 11387, 11397, 11401, 11405, 11408, 11414, 11418, 11421, 11422, 11424,
11427,
11429, 11430, 11377, 11379, 11380, 11385, 11386, 11388, 11390, 11391, 11392,
11394,
11395, 11396, 11398, 11399, 11400, 11402, 11403, 11404, 11406, 11409, 11410,
11411,
11412, 11415, 11416, 11417, 11419, 11423, 11425, 11428, 11431, 11432, 11433,
11434,
11438, 11442, 11443, 11447, 11449, 11450, 11452, 11456, 11459, 11463, 11464,
11467,
11468, 11472, 11477, 11480, 11481, 11482, 11483, 11484, 11488, 11489, 11493,
11498,
11502, 11504, 11505, 11506, 11507, 11508, 11509, 11512, 11513, 11514, 11517,
11520,
11522, 11523, 11524, 11526, 11528, 11532, 11534, 11435, 11439, 11444, 11445,
11446,
11448, 11451, 11453, 11457, 11458, 11460, 11461, 11462, 11465, 11466, 11469,
11473,
11474, 11475, 11476, 11478, 11479, 11485, 11486, 11487, 11490, 11494, 11497,
11499,
11500, 11501, 11503, 11510, 11511, 11515, 11518, 11519, 11521, 11525, 11527,
11529,
11535, 11539, 11540, 11541, 11548, 11550, 11551, 11552, 11555, 11556, 11557,
11560,
11561, 11562, 11568, 11569, 11571, 11572, 11573, 11577, 11536, 11542, 11545,
11546,
11547, 11549, 11553, 11554, 11563, 11566, 11570, 11574, 11578, 11585, 11586,
11590,
11591, 11592, 11595, 11596, 11597, 11600, 11579, 11582, 11587, 11593, 11594,
11598,
11601, 11602, 11606, 11607, 11608, 11609, 11616, 11618, 11622, 11626, 11628,
11632,
11633, 11603, 11610, 11613, 11614, 11615, 11617, 11619, 11623, 11627, 11629,
11634,
11635, 11640, 11641, 11642, 11643, 11647, 11648, 11654, 11655, 11656, 11657,
11658,
98
CA 02528853 2005-12-23
11636, 11639, 11644, 11645, 11646, 11649, 11652, 11653, 11659, 11663, 11664,
11665,
11666, 11670, 11671, 11672, 11673, 11677, 11681, 11687, 11690, 11694, 11696,
11697,
11701, 11705, 11709, 11713, 11719, 11723, 11729, 11733, 11739, 11744, 11745,
11748,
11749, 11751, 11755, 11759, 11763, 11770, 11772, 11774, 11776, 11778, 11782,
11785,
11660, 11667, 11674, 11675, 11676, 11678, 11682, 11685, 11686, 11688, 11689,
11691,
11692, 11693, 11695, 11698, 11699, 11700, 11702, 11706, 11710, 11716, 11720,
11724,
11725, 11727, 11728, 11730, 11731, 11732, 11734, 11735, 11736, 11737, 11738,
11740,
11743, 11746, 11747, 11750, 11752, 11756, 11761, 11762, 11764, 11765, 11767,
11768,
11769, 11773, 11775, 11777, 11779, 11783, 11784, 11786, 11790, 11791, 11792,
11787,
11793, 11796, 11800, 11802, 11803, 11805, 11810, 11818, 11819, 11823, 11824,
11827,
11828, 11829, 11833, 11836, 11837, 11839, 11840, 11847, 11850, 11852, 11857,
11860,
11864, 11866, 11797, 11801, 11804, 11806, 11809, 11811, 11814, 11815, 11817,
11820,
11825, 11830, 11835, 11841, 11844, 11845, 11846, 11848, 11851, 11853, 11856,
11858,
11861, 11862, 11863, 11865, 11867, 11871, 11876, 11878, 11882, 11888, 11891,
11894,
11868, 11872, 11875, 11877, 11879, 11883, 11886, 11887, 11889, 11890, 11892,
11893,
11895, 11898, 11902, 11903, 11904, 11908, 11909, 11911, 11912, 11913, 11914,
11915, '
11917, 11918, 11922, 11926, 11927, 11930, 11932, 11933, 11937, 11941, 11942,
11946,
11947, 11948, 11949, 11950, 11951, 11952, 11955, 11959, 11968, 11972, 11976,
11978,
11979, 11980, 11981, 11986, 11988, 11989, 11991, 11992, 11995, 11997, 11998,
12001,
12002, 12004, 12005, 11899, 11905, 11910, 11916, 11919, 11923, 11924, 11925,
11928,
11929, 11931, 11934, 11938, 11943, 11953, 11954, 11956, 11960, 11963, 11964,
11967,
11969, 11973, 11977, 11982, 11985, 11987, 11990, 11993, 11994, 11996, 11999,
12000,
12003, 12006, 12011, 12015, 12016, 12017, 12021, 12022, 12023, 12024, 12025,
12026,
12027, 12031, 12034, 12042, 12043, 12045, 12049, 12050, 12051, 12054, 12062,
12063,
12064, 12066, 12067, 12068, 12070, 12072, 12076, 12082, 12085, 12086, 12089,
12090,
12093, 12094, 12095, 12096, 12097, 12098, 12101, 12103, 12107, 12108, 12111,
12112,
12114, 12118, 12119, 12120, 12128, 12007, 12010, 12012, 12020, 12028, 12032,
12033,
12035, 12038, 12039, 12044, 12046, 12052, 12055, 12056, 12057, 12058, 12059,
12060,
12061, 12065, 12069, 12071, 12073, 12077, 12080, 12081, 12084, 12087, 12088,
12091,
99
CA 02528853 2005-12-23
12099, 12100, 12102, 12104, 12109, 12110, 12113, 12115, 12121, 12122, 12123,
12124,
12125, 12127, 12129, 12130, 12135, 12136, 12141, 12144, 12147, 12148, 12149,
12153,
12159, 12160, 12131, 12134, 12137, 12138, 12139, 12140, 12142, 12143, 12145,
12146,
12150, 12154, 12157, 12158, 12161, 12162, 12163, 12164, 12168, 12171, 12176,
12181,
12187, 12190, 12194, 12196, 12197, 12199, 12201, 12202, 12209, 12213, 12216,
12220,
12221, 12223, 12225, 12226, 12231, 12233, 12239, 12243, 12244, 12250, 12254,
12256,
12257, 12259, 12266, 12270, 12271, 12272, 12274, 12276, 12277, 12278, 12280,
12284,
12285, 12288, 12289, 12290, 12291, 12295, 12296, 12302, 12308, 12313, 12317,
12321,
12322, 12323, 12324, 12325, 12327, 12328, 12330, 12331, 12332, 12336, 12337,
12339,
12343, 12346, 12349, 12353, 12354, 12355, 12359, 12361, 12363, 12367, 12369,
12370,
12372, 12376, 12377, 12379, 12380, 12381, 12382, 12383, 12385, 12389, 12393,
12398,
12400, 12402, 12403, 12406, 12408, 12409, 12410, 12412, 12415, 12417, 12419,
12422,
12424, 12429, 12430, 12432, 12433, 12435, 12436, 12437, 12441, 12445, 12448,
12449,
12454, 12458, 12459, 12460, 12165, 12169, 12170, 12172, 12175, 12177, 12180,
12182,
12185, 12186,12188, 12191, 12195, 12198, 12200, 12203, 12206, 12207, 12208,
12210,
12214, 12217, 12222, 12224, 12227, 12228, 12229, 12230, 12232, 12234, 12237,
12238,
12240, 12241, 12242, 12245, 12248, 12249, 12251, 12255, 12258, 12260, 12262,
12263,
12264, 12265, 12267, 12268, 12269, 12273, 12279, 12281, 12286, 12287, 12292,
12297,
12300, 12303, 12306, 12307, 12309, 12312, 12314, 12318, 12326, 12333, 12338,
12340,
12341, 12342, 12345, 12347, 12348, 12352, 12356, 12360, 12362, 12364, 12371,
12373,
12378, 12386, 12390, 12394, 12397, 12401, 12404, 12405, 12411, 12413, 12418,
12420,
12423, 12425, 12428, 12438, 12439, 12440, 12442, 12443, 12446, 12450, 12453,
12455,
12461, 12465, 12467, 12468, 12472, 12474, 12477, 12480, 12484, 12486, 12488,
12489,
12490, 12493, 12495, 12499, 12501, 12504, 12506, 12462, 12469, 12475, 12478,'
12481,
12483, 12485, 12487, 12492, 12494, 12496, 12497, 12498, 12500, 12502, 12505,
12507,
12511, 12517, 12520, 12522, 12523, 12524, 12528, 12530, 12534, 12535, 12536,
12537,
12541, 12542, 12547, 12550, 12551, 12552, 12554, 12555, 12560, 12564, 12565,
12569,
12571, 12574, 12579, 12581, 12586, 12590, 12592, 12593, 12594, 12596, 12600,
12601,
12602, 12604, 12610, 12613, 12614, 12615, 12616, 12620, 12626, 12630, 12508,
12512,
100
101
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CA 02528853 2005-12-23
13057, 13062, 13064, 13065, 13066, 13067, 13072, 13073, 13076, 13078, 13082,
13083,
13085, 13087, 13088, 13089, 13092, 13096, 13100, 13101, 13102, 13105, 13108,
13109,
13110, 13114, 13117, 13118, 13124, 13129, 13131, 13132, 13138, 13144, 13146,
13147,
13148, 13152, 13156, 13159, 13164, 13167, 13168, 13170, 13171, 13172, 13173,
13174,
13184, 13185, 13006, 13010, 13014, 13018, 13020, 13021, 13022, 13023, 13024,
13026,
13031, 13035, 13036, 13038, 13039, 13041, 13045, 13050, 13054, 13058, 13061,
13063,
13068, 13069, 13070, 13071, 13074, 13075, 13077, 13079, 13084, 13086, 13090,
13091,
13093, 13097, 13104, 13107, 13111, 13115, 13119, 13121, 13122, 13123, 13125,
13126,
13127, 13128, 13130, 13133, 13137, 13139, 13142, 13143, 13145, 13149, 13153,
13157,
13158, 13160, 13163, 13165, 13166, 13169, 13175, 13176, 13177, 13178, 13179,
13180,
13181, 13182, 13183, 13186, 13189, 13190, 13191, 13195, 13196, 13197, 13199,
13200,
13204, 13206, 13192, 13198, 13201, 13207, 13212, 13213, 13226, 13228, 13231,
13235,
13240, 13245, 13247, 13248, 13250, 13254, 13256, 13258, 13264, 13265, 13268,
13269,
13273, 13274, 13277, 13279, 13280, 13281, 13286, 13208, 13211, 13214, 13217,
13220,
13222, 13224, 13225, 13227, 13229, 13232, 13233, 13234, 13237, 13239, 13241,
13242,
13244, 13246, 13249, 13251, 13255, 13257, 13259, 13262, 13263, 13266, 13267,
13270,
13275, 13276, 13282, 13285, 13641, 13645, 13646, 13649, 13650, 13652, 13653,
13654,
13655, 13658, 13659, 13642, 13647, 13648, 13656, 13660, 13661, 13664, 13669,
13670,
13671, 13672, 13673, 13675, 13676, 13677, 13681, 13683, 13685, 13687, 13689,
13691,
13693, 13696, 13665, 13668, 13674, 13678, 13682, 13684, 13686, 13688, 13690,
13692,
13694, 13695, 13697, 13701, 13702, 13703, 13709, 13713, 13714, 13715, 13719,
13721,
13722, 13726, 13728, 13732, 13735, 13740, 13698, 13704, 13705, 13706, 13707,
13708,
13710, 13716, 13717, 13718, 13720, 13723, 13724, 13725, 13727, 13729, 13733,
13734,
13736, 13739, 13741, 13744, 13745, 13749, 13751, 13752, 13753, 13754, 13758,
13762,
13765, 13766, 13768, 13772, 13773, 13774, 13777, 13778, 13779, 13783, 13785,
13790,
13791, 13797, 13802, 13803, 13805, 13806, 13807, 13809, 13812, 13814, 13816,
13817,
13818, 13819, 13820, 13823, 13824, 13826, 13828, 13746, 13755, 13759, 13763,
13764,
13767, 13769, 13775, 13776, 13780, 13784, 13786, 13788, 13789, 13792, 13795,
13796,
13798, 13800, 13804, 13808, 13813, 13815, 13821, 13822, 13829, 13832, 13837,
13841,
102
CA 02528853 2005-12-23
13844, 13845, 13846, 13849, 13854, 13855, 13856, 13857, 13861, 13865, 13869,
13871,
13875, 1387% 13880, 13833, 13836, 13838, 13843, 13847, 13850, 13853, 13858,
13862,
13866, 13870, 13872, 13876, 13881, 13882, 13883, 13889, 13894, 13902, 13903,
13910,
13911, 13914, 13915, 13916, 13928, 13929, 13930, 13931, 13935, 13936, 13937,
13944,
13947, 13948, 13950, 13960, 13961, 13962, 13964, 13965, 13966, 13968, 13970,
13971,
13972, 13976, 13978, 13884, 13887, 13890, 13893, 13895, 13898, 13900, 13904,
13905,
13906, 13907, 13908, 13909, 13912, 13913, 13917, 13920, 13921, 13922, 13923,
13924,
13925, 13926, 13932, 13938, 13941, 13942, 13943, 13945, 13946, 13949, 13953,
13954,
13956, 13957, 13958, 13959, 13963, 13967, 13969, 13974, 13975, 13977, 13980,
13981,
13988, 13989, 13990, 13992, 13994,13995, 13999, 13982, 13985, 13986, 13987,
13991,
13993, 13996, 14000, 14001, 14002, 14003, 14004, 14009, 14014, 14017, 14020,
14024,
14026, 14029, 14035, 14036, 14040, 14043, 14045, 14051, 14057, 14058, 14059,
14063,
14065, 14066, 14067, 14068, 14069, 14070, 14071, 14073, 14074, 14078, 14079,
14080,
14081, 14082, 14083, 14084, 14085, 14087, 14094, 14095, 14100, 14108, 14109,
14113,
14114, 14118, 14120, 14121, 14122, 14130, 14133, 14138, 14141, 14142, 14143,
14147,
14005, 14008, 14010, 14013, 14015, 14018, 14021, 14025, 14027, 14028, 14030,
14033,
14034, 14037, 14038, 14039, 14041, 14042, 14044, 14046, 14049, 14050, 14052,
14055, .
14056, 14060, 14064, 14072, 14077, 14086, 14088, 14089, 14090, 14091, 14092,
14093,
14096, 14097, 14098, 14099, 14101, 14102, 14103, 14104, 14105, 14106, 14107,
14110,
14115, 14123, 14126, 14127, 14132, 14134, 14135, 14136, 14137, 14139, 14140,
14144,
14145, 14148, 14151, 14155, 14157, 14161, 14166, 14171, 14175, 14183, 14187,
14190,
14192, 14194, 14195, 14196, 14200, 14201, 14152, 14156, 14158, 14162, 14165,
14167,
14170, 14172, 14176, 14179, 14180, 14182, 14184, 14185, 14186, 14188, 14189,
14191,
14193, 14197, 14198, 14199, 14202, 14203, 14204, 14210, 14211, 14216, 14217,
14223,
14226, 14228, 14229, 14230, 14233, 14235, 14239, 14241, 14243, 14244, 14250,
14254,
14255, 14256, 14257, 14260, 14261, 14262, 14267, 14268, 14269, 14273, 14274,
14277,
14281, 14284, 14286, 14289, 14292, 14293, 14297, 14299, 14301, 14304, 14305,
14306,
14312, 14316, 14320, 14322, 14324, 14328, 14329, 14331, 14337, 14342, 14344,
14346,
14347, 14349, 14350, 14353, 14356, 14361, 14363, 14364, 14365, 14366, 14205,
14208,
103
CA 02528853 2005-12-23
14212, 14215, 14218, 14221, 14224, 14225, 14227, 14231, 14236, 14240, 14242,
14245,
14246, 14247, 14251, 14263, 14266, 14270, 14278, 14282, 14283, 14285, 14287,
14290,
14291, 14294, 14298, 14300, 14302, 14303, 14307, 14310, 14313, 14317, 14323,
14325,
14327, 14330, 14334, 14338, 14340, 14341, 14345, 14348, 14351, 14352, 14354,
14355,
14357, 14358, 14359, 14360, 14367, 14368, 14372, 14373, 14375, 14386, 14369,
14374,
14376,14379, 14380, 14381,14382, 14383,14384, and SEQ IDNO:14385.
Step e) of the method, namely the detection of the specific amplificates
indicative of the
methylation status of one or more CpG positions according to SEQ II) NOS:1 to
SEQ ID
NO:64 is carried out by means of real-time detection methods as descn'bed
above, and wherein
the sequence of said hybridization probes is selected from the group
consisting of SEQ ID
NOS:2753, 2756, 2758, 2761, 2766, 2770, 2774, 2778, 2799, 2807, 2813, 2819,
2822, 2826,
2848, 2857, 2900, 2906, 2908, 2910, 2913, 2921, 2925, 2940, 2943, 2952, 2964,
2983, 2986,
3009, 3012, 3015, 3024, 3030, 3035, 3046, 3058, 3062, 3067, 3070, 3074, 3077,
3079, 3082,
3087, 3095, 3099, 3102, 3106, 3112, 3120, 3125, 3129, 3132, 3141, 3143, 3154,
3156, 3158,
3161, 3177, 3188, 3191, 3193, 3200, 3208, 3211, 3220, 3231, 3241, 3251, 3254,
3258, 3263,
3272, 3277, 3288, 3294, 3296, 3299, 3303, 3317, 3323, 3326, 3329, 3336, 3341,
3362, 3366,
3370, 3375, 3379, 3382, 3387, 3394, 3398, 3405, 3407, 3410, 3412, 3416, 3422,
3425, 3430,
3449, 3451, 3457, 3460, 3465, 3468, 3486, 3498, 3502, 3507, 3523, 3532, 3539,
3546, 3550,
3567, 3572, 3579, 3582, 3610, 3620, 3628, 3648, 3659, 3664, 3672, 3675, 3680,
3682, 3685,
3687, 3699, 3712, 3716, 3720, 3734, 3751, 3763, 3766, 3769, 3783, 3789, 3795,
3811, 3816,
3825, 3830, 3835, 3841, 3843, 3846, 3849, 3855, 3859, 3879, 3882, 3910, 3912,
3913, 3918,
3925, 3929, 3932, 3946, 3953, 3956, 3967, 3969, 3973, 3977, 3989, 3992, 3996,
3999, 4002,
4008, 4014, 4026, 4030, 4036, 4041, 4044, 4053, 4057, 4060, 4062, 4081, 4091,
4097, 4103,
4107, 4118, 4126, 4140, 4143, 4147, 4155, 4158, 4164, 4175, 4178, 4183, 4187,
4193, 4200,
4203, 4210, 4212, 4215, 4222, 4227, 4230, 4235, 4240, 4250, 4253, 4264, 4278,
4294, 4312,
4314, 4320, 4329, 4339, 4346, 4356, 4363, 4380, 4402, 4404, 4414, 4419, 4421,
4431, 4439,
4445, 4449, 4459, 4468, 4483, 4493, 4500, 4513, 4526, 4527, 4532, 4544, 4550,
4552, 4566,
4577, 4591, 4594, 4604, 4608, 4623, 4625, 4629, 4637, 4651, 4654, 4657, 4673,
4679, 4682,
104
CA 02528853 2005-12-23
4692, 4698, 4710, 4712, 4713, 4723, 4725, 4729, 4734, 4740, 4743, 4747, 4752,
4757, 4762,
4765, 4775, 4781, 4796, 4799, 4803, 4809, 4826, 4831, 4863, 4866, 4880, 4888,
4914, 4916,
4931, 4933, 4939, 4955, 4959, 4962, 4965, 4994, 5001, 5008, 5012, 5023, 5029,
5035, 5046,
5059, 5063, 5068, 5071, 5079, 5082, 5085, 5089, 5092, 5095, 5098, 5102, 5108,
5113, 5118,
5122, 5127, 5130, 5133, 5138, 5141, 5146, 5152, 5154, 5178, 5192, 5197, 5206,
5213, 5228,
5236, 5243, 5258, 5267, 5275, 5277, 5283, 5294, 5298, 5302, 5305, 5320, 5325,
5328, 5335,
5345, 5350, 5357, 5374, 5381, 5384, 5386, 5390, 5397, 5402, 5406, 5411, 5423,
5430, 5439,
5453, 5463, 5473, 5476, 5479, 5483, 5489, 5514, 5520, 5523, 5530, 5547, 5584,
5590, 5607,
5610, 5613, 5617, 5621, 5631, 5636, 5641, 5653, 5655, 5658, 5666, 5669, 5675,
5685,. 5692,
5695, 5703, 5706, 5716, 5719, 5726, 5738, 5746, 5749, 5752, 5755, 5757, 5761,
5765, 5772,
5784, 5799, 5805, 5815, 5819, 5829, 5851, 5869, 5901, 5908, 5911, 5914, 5917,
5925, 5941,
5951, 5967, 5974, 5979, 5982, 5985, S989, 5995, 6022, 6029, 6044, 6047, 6052,
6057, 6061,
6069, 6071, 6080, 6082, 6087, 6092, 6095, 6102, 6104, 6107, 6109, 6113, 6124,
6129, 6140;
6147, 6158, 6165, 6179, 6185, 6203, 6209, 6217, 6228, 6249, 6263, 6267, 6279,
6290, 6332,
6339, 6343, 6346, 6352, 6363, 6366, 6370, 6380, 6385, 6391, 6396, 6408, 6430,
6439, 6454,
6456, 6477, 6483, 6488, 6495, 6509, 6517, 6520, 6530, 6534, 6555, 6557, 6559,
6569, 6574,
6581, 6593, 6616, 6622, 6626, 6636, 6637, 6648, 6654, 6667, 6673, 6684, 6724,
6729, 6733,
6737, 6743, 6750, 6753, 6763, 6766, 6768, 6771, 6776, 6780, 6788, 6793, 6808,
6813, 6820,
6823, 6826, 6846, 6856, 6864, 6867, 6878, 6885, 6903, 6914, 6919, 6923, 6926,
6938, 6941,
6978, 6982, 6985, 6988, 6994, 6997, 7000, 7004, 7008, 7015, 7019, 7021, 7069,
7079, 7099,
7104, 7117, 7120, 7145, 7151, 7153, 7162, 7164, 7169, 7201, 7209, 7234, 7238,
7252, 7286,
7288, 7300, 7305, 7313, 7323, 7327, 7338, 7343, 7346, 7352, 7377, 7380, 7396,
7399, 7406,
7412, 7414, 7418, 7424, 7429, 7437, 7441, 7443, 7458, 7461, 7469, 7473, 7491,
7502, 7504,
7514, 7531, 7551, 7553, 7557, 7562, 7564, 7569, 7572, 7592, 7602, 7605, 7612,
7625, 7630,
7632, 7634, 7648, 7653, 7664, 7668, 7693, 7696, 7706, 7763, 7766, 7769, 7772,
7777, 7780,
7787, 7790, 7801, 7804, 7806, 7826, 7831, 7850, 7859, 7862, 7867, 7876, 7883,
7887, 7891,
7893, 7895, 7898, 7901, 7906, 7908, 7911, 7915, 7924, 7927, 7930, 7935, 7959,
7961, 7966,
7975, 7978, 7984, 7994, 7998, 8002, 8014, 8029, 8032, 8037, 8041, 8045, 8048,
8058, 8063,
8086, 8092, 8097, 8106, 8109, 8120, 8140, 8141, 8159, 8162, 8172, 8196, 8205,
8211, 8215,
105
CA 02528853 2005-12-23
8219, 8226, 8234, 8241, 8249, 8262, 8265, 8272, 8276, 8279, 8283, 8290, 8300,
8308, 8320,
8338, 8355, 8358, 8368, 8380,8386, 8389, 8393, 8396, 8400, 8404, 8408, 8417,
8421, 8427,
8430, 8439, 8441, 8443, 8453, 8459, 8471, 8483, 8495, 8507, 8509, 8525, 8538,
8540, 8543,
8549, 8553, 8559, 8561, 8573, 8579, 8584, 8590, 8593, 8605, 8611, 8622, 8626,
8656, 8664,
8721, 8724, 8731, 8740, 8743, 8746, 8765, 8774, 8781, 8792, 8795, 8811, 8817,
8822, 8825,
8834, 8839, 8849, 8858, 8860, 8862, 8875, 8886, 8910, 8915, 8919, 8922, 8926,
8940, 8947,
8956, 8959, 8973, 8982, 8986, 8991, 8996, 9000, 9015, 9027, 9031, 9040, 9057,
9059, 9062,
9065, 9073, 9076, 9101, 9115, 9117, 9120, 9132, 9145, 9161, 9170, 9179, 9196,
9210, 9213,
9225, 9236, 9242, 9245, 9250, 9255, 9259, 9262, 9268, 9272, 9294, 9299, 9306,
9315, 9330,
9341; 9343, 9346, 9350, 9359, 9362, 9366, 9369, 9373, 9381, 9387, 9392, 9396,
9404, 9427,
9442, 9463, 9472, 9476, 9481, 9484, 9486, 9491, 9507, 9510, 9531, 9540, 9553,
9562, 9568,
9572, 9575, 9582, 9590, 9597, 9600, 9610, 9616, 9620, 9629, 9636, 9642, 9648,
9655, 9669,
9674, 9678, 9681, 9685, 9689, 9696, 9704, 9709, 9725, 9728, 9731, 9736, 9740,
9743, 9752,
9770, 9774, 9779, 9785, 9799, 9802, 9814, 9824, 9829, 9834, 9874, 9884, 9888,
9906, 9910,
9927, 9930, 9951, 9965, 9971, 9977, 10024, 10029, 10039, 10052, 10063, 10066,
10073,
10076, 10085, 10108, 10115, 10128, 10136, 10146, 10158, 10162, 10168, 10171,
10180,
10185, 10208, 10217, 10238, 10246, 10250, 10281, 10284, 10289, 10294, 10296,
10303,
10316, 10324, 10332, 10334, 10339, 10353, 10364, 10368, 10371, 10389, 10393,
10396,
10397, 10400, 10405, 10409, 10421, 10424, 10436, 10441, 10450, 10458, 10472,
10474,
10479, _ 10482, 10486, 10496, 10505, 10512, 10527, 10549, 10555, 10562, 10567,
10570,
10575, 10585, 10587, 10591, 10595, 10599, 10608, 10615, 10620, 10623, 10629,
10636,
10677, 10680, 10686, 10692, 10695, 10698, 10701, 10706, 10708, 10715, 10731,
10736,
10753, 10756, 10766, 10771, 10774, 10777, 10785, 10787, 10795, 10797, 10803,
10810,
10825, 10828, 10832, 10836, 10852, 10855, 10867, 10876, 10887, 10891, 10895,
10898,
10913, 10918, 10921, 10923, 10936, 10940, 10949, 10961, 10964, 10974, 10983,
10990,
10994, 11003, 11008, 11011, 11018, 11024, 11035, 11038, 11057, 11095, 11378,
11389,
11393, 11407, 11413, 11420, 11426, 11436, 11440, 11454, 11470, 11491, 11495,
11530,
11437, 11441, 11455, 11471, 11492, 11496, 11516, 11531, 11533, 11537, 11543,
11558,
11564, 11575, 11538, 11544, 11559, 11565, 11567, 11576, 11580, 11583, 11588,
11581,
106
CA 02528853 2005-12-23
11584, 11589, 11599, 11634, 11604, 11611, 11620, 11624, 11630, 11605, 11612,
11621,
11625, 11631, 11637, 11650, 11638, 11651, 11661, 11668, 11679, 11683, 11703,
11707,
11711, 11714, 11717, 11721, 11726, 11741, 11753, 11757, 11766, 11771, 11780,
11662,
11669, 11680, 11684, 11704, 11708, 11712, 11715, 11718, 11722, 11742, 11754,
11758,
11760, 11781, 11788, 11794, 11789, 11795, 11798, 11807, 11812, 11816, 11821,
11831,
11842, 11854, 11799, 11808, 11813, 11822, 11826, 11832, 11834, 11838, 11843,
11849,
11855, 11859, 11869, 11873, 11880, 11884, 11896, 11870, 11874, 11881, 11885,
11897,
11900, 11906, 11920, 11935, 11939, 11944, 11957, 11961, 11965, 11970, 11974,
11983,
11901, 11907, 11921, 11936, 11940, 11945, 11958, 11962, 11966, 11971, 11975,
11984,
12008, 12013, 12018, 12029, 12036, 12040, 12047, 12074, 12078, 12083, 12105,
12116,
12009, 12014, 12019, 12030, 12037, 12041, 12048, 12053, 12075, 12079, 12036,
12092,
12106, 12117, 12126, 12132, 12151, 12155, 12133, 12152, 12156, 12166, 12173,
12178,
12183, 12189, 12192, 12204, 12211, 12218y 12235, 12246, 12252, 12282, 12293,
12298,
12301, 12304, 12310, 12315, 12319, 12334, 12350, 12357, 12365, 12374, 12387,
12391,
12395, 12416, 12426, 12444, 12451, 12456, 12167, 12174, 12179, 12184, 12193,
12205,
12212, 12215, 12219, 12236, 12247, 12253, 12261, 12275, 12283, 12294, 12299,
12305,
12311, 12316, 12320, 12329, 12335, 12344, 12351, 12358, 12366, 12368, 12375,
12384,
12388, 12392, 12396, 12399, 12407, 12414, 12421, 12427, 12431, 12434, 12447,
12452,
12457, 12463, 12470, 12473, 12482, 12464, 12466, 12471, 12476, 12479, 12491,
12503,
12509, 12513, 12516, 12514, 12532, 12539, 12544, 12557, 12562, 12567, 12576,
12583,
12588, 12591, 12606, 12612, 12622, 12628, 12510, 12514, 12519, 12526, 12527,
12533,
12540, 12545, 12558, 12563, 12568, 12577, 12580, 12584, 12589, 12598, 12607,
12619,
12623, 12629, 12633, 12642, 12645, 12650, 12656, 12668, 12674, 12676, 12697,
12707,
12722, 12734, 12741, 12759, 12773, 12779, 12783, 12802, 12806, 12810, 12819,
12829,
12634, 12646, 12651, 12657, 12659, 12669, 12698, 12708, 12711, 12723, 12707,
12727,
12735, 12742, 12774, 12780, 12784, 12803, 12807, 12811, 12820, 12830, 12847,
12852,
12859, 12864, 12874, 12905, 12915, 12925, 12930, 12934, 12940, 12944, 12946,
12955,
12848, 12853, 12856, 12860, 12865, 12875, 12897, 12906, 12916, 12922, 12926,
12931,
12935, 12941, 12930, 12947, 12952, 12956, 12963, 12979, 12964, 12980, 12984,
13003,
107
CA 02528853 2005-12-23
12985, 12988, 12990, 12994, 13004, 13007, 13011, 13015, 13027, 13046, 13055,
13059,
13080, 13094, 13098, 13112, 13120, 13134, 13140, 13150, 13154, 13161, 13187,
13008,
13012, 13016, 13028, 13047, 13056, 13060, 13081, 13095, 13099, 13103, 13106,
13113,
13116, 13135, 13136, 13141, 13151, 13155, 13162, 13188, 13193, 13202, 13194,
13203,
13205, 13209, 13215, 13218, 13236, 13252, 13260, 13271, 13283, 13210, 13216,
13219,
13221, 13223, 13230, 13238, 13243, 13253, 13261, 13272, 13278, 13284, 13643,
13644,
13651, 13657, 13662, 13663, 13666, 13679, 13667, 13680, 13699, 13711, 13730,
13737,
13742, 13700, 13712, 13731, 13738, 13743, 13747, 13756, 13760, 13781, 13793,
13801,
13810, 13830, 13748, 13750, 13757, 13761, 13770, 13771, 13782, 13787, 13794,
13799,
13811, 13825, 13827, 13831, 13834, 13839, 13851, 13859, 13863, 13867, 13873,
13877,
13835, 13840, 13842, 13848, 13852, 13860, 13864, 13868, 13874, 13878, 13885,
13886,
13891, 13896, 13901, 13918, 13927, 13933, 13939, 13951, 13886, 13888, 13892,
13897,
13899, 13901, 13919, 13934, 13940, 13952, 13955, 13973, 13979, 13983, 13997,
13984,
13998, 14006, 14011, 14019, 14022, 14031, 14047, 14053, 14061, 14075, 14111,
14116,
14124, 14149, 14007, 14012, 14016, 14023, 14032, 14048, 14054, 14062, 14076,
14112,
14117, 14119, 14125, 14128, 14129, 14131, 14146, 14150, 14153, 14159, 14163,
14168,
14173, 14177, 14154, 14160, 14164, 14169, 14174, 14178, 14181, 14206, 14213,
14219,
14237, 14248, 14252, 14258, 14264, 14271, 14275, 14279, 14295, 14308, 14311,
14314,
14318, 14332, 14335, 14339, 14343, 14207, 14209, 14214, 14220, 14222, 14232,
14234,
14238, 14249, 14253, 14259, 14265, 14272, 14276, 14280, 14288, 14296, 14309,
14315,
14319, 14321, 14326, 14333, 14336, 14362, 14370,14377,14371 arul SEQ ID
NO:14378.
For each genomic sequence listed, the foliowing oligonucleotides as detailed
in the
sequence listing may be used for a combined 1VISP-RealTime analysis:
Taqman Raal time single probe detection assays:
SEQ ID NO: 41
left Primer: 2751, 2754, 2757, 2759, 2767, 2768, 2772, 2776, 2779, 2784, 2785,
2786,
2789, 2791, 2792, 2794, 2797, 2798, 2801, 2805, 2806, 2809, 2811, 2815, 2817,
2824, 2830,
2832, 2833, 2834, 2838, 2840, 2843, 2846, 2850, 2851, 2853, 2855, 2859, 2866,
2867, 2870,
2871, 2876, 2878, 2881, 2885, 2886, 2892, 2893, 2895, 2896, 2898, 2904, 2912,
2915, 2917,
108
CA 02528853 2005-12-23
2919, 2922, 2923, 2929, 2931, 2939, 2941, 2944, 2946, 2948, 2950, 2955, 2956,
2957, 2960,
2962, 2965, 2966, 2967, 2968, 2969, 2971, 2975, 2976, 2977, 2978, 2979, 2980,
2981, 2984,
2988, 2989, 2990, 2992, 2994, 2997, 2999, 3001, 3002, 3004, 3006, 3007, 3008,
3010, 3013,
3014, 3016, 3017, 3018, 3020, 3022, 3025,
right Primer. 2752, 2755, 2760, 2762, 2763, 2764, 2765, 2769, 2771, 2773,
2775, 2777,
2780, 2781, 2782, 2783, 2787, 2788, 2790, 2793, 2795, 2796, 2800, 2802, 2803,
2804, 2808,
2810, 2812, 2814, 2816, 2818, 2820, 2821, 2823, 2825, 2827, 2828, 2829, 2831,
2835, 2836,
2837, 2839, 2841, 2842, 2844, 2845, 2847, 2849, 2852, 2854, 2856, 2858, 2860,
2861, 2862,
2863, 2864, 2865, 2868, 2869, 2872, 2873, 2874, 2875, 2877, 2879, 2880, 2882,
2883, 2884,
2887, 2888, 2884, 2890, 2891, 2894, 2897, 2899, 2901, 2902, 2903, 2905, 2907,
2909, 2911,
2914, 2916, 2918, 2920, 2924, 2926, 2927, 2928, 2930, 2932, 2933, 2934, 2935,
2936, 2937,
2938, 2942, 2945, 2947, 2949, 2951, 2953, 2954, 2958, 2959, 2961, 2963, 2970,
2972, 2973,
2974, 2982, 2985, 2987, 2991, 2993, 2995, 2996, 2998, 3000, 3003, 3005, 3011,
3019, 3021,
3023, 3026, 3027,
Detection: 2753, 2756, 2 758, 2761, 2766, 2770, 2774, 2778, 2799, 2807, 2813,
2819,
2822, 2826, 2848, 2857, 2900, 2906, 2908, 2910, 2913, 2921, 2925, 2940, 2943,
2952, 2964,
2983, 2986, 3009, 3012, 3015, 3024,
SEQ ID NO: 5
left Primer: 3028, 3032, 3033, 3036, 3037, 3038, 3039, 3041, 3042, 3043, 3044,
3047,
3048, 3049, 3052, 3055, 3059, 3061, 3064, 3065, 3068, 3069, 3071, 3072, 3075,
3076, 3080,
3083, 3084, 3085, 3086, 3091, 3093, 3096, 3097, 3100, 3104, 3109, 3110, 3113,
3115, 3117,
3118, 3123, 3126, 3127, 3130, 3134, 3135, 3136, 3138, 3139, 3144, 3146, 3147,
3149, 3150,
3155,
right Primer: 3029, 3031, 3034, 3040, 3045, 3050, 3051, 3053, 3054, 3056,
3057, 3060,
3063, 3066, 3073, 3078, 3081, 3088, 3089, 3090, 3092, 3094, 3098, 3101, 3103,
3105, 3107,
3108, 3111, 3114, 3116, 3119, 3121, 3122, 3124, 3128, 3131, 3133, 3137, 3140,
3142, 3145,
3148, 3151, 3152, 3153, 3157,
petection: 3030, 3035, 3 046, 3058, 3062, 3067, 3070, 3074, 3077, 3079, 3082,
3087,
109
CA 02528853 2005-12-23
3095, 3099, 3102, 3106, 3112, 3120, 3125, 3129, 3132, 3141, 3143, 3154, 3156,
3158,
SEQ ID NO: 6
left Primer: 3159, 3165, 3166, 3167, 3170, 3171, 3172, 3174, 3175, 3176, 3179,
3180,
3181, 3184, 3185, 3186, 3192, 3194, 3198, 3201, 3202, 3203, 3204, 3206, 3209,
3212, 3213,
3215, 3216, 3217, 3218, 3221, 3224, 3226, 3228, 3229, 3230, 3232, 3233, 3234,
3235, 3237,
3238, 3239, 3245, 3247, 3248, 3249,
right Primer: 3160, 3162, 3163, 3164, 3168, 3169, 3173, 3178, 3182, 3183,
3187, 3189,
3190, 3195, 3196, 3197, 3199, 3205, 3207, 3210, 3214, 3219, 3222, 3223, 3225,
3227, 3236,
3240, 3242, 3243, 3244, 3246, 3250,
Detection: 3161, 3177, 3188, 3191, 3193, 3200, 3208, 3211, 3220, 3231, 3241,
3251,
SEQ ID NO: 8
left Primer: 3252, 3256, 3261, 3264, 3268, 3269, 3270, 3273, 3275, 3279, 3280,
3281,
3282, 3283, 3284, 3285, 3286, 3290, 3292, 3295, 3297, 3301, 3304, 3305, 3306,
3307, 3308,.
3310, 3311, 3312, 3315, 3319, 3320, 3324, 3327, 3331, 3333, 3334, 3339, 3348,
3351, 3354,.
3358, 3360, 3364, 3367, 3368, 3374,3376,
right Primer. 3253, 3255, 3257, 3259, 3260, 3262, 3265, 3266, 3267, 3271,
3274, 3276,
3278, 3287, 3289, 3291, 3293, 3298, 3300, 3302, 3309, 3313, 3314, 3316, 3318,
3321, 3322,
3325, 3328, 3330, 3332, 3335, 3337, 3338, 3340, 3342, 3343, 3344, 3345, 3346,
3347, 3349,
3350, 3352, 3353, 3355, 3356, 3357, 3359, 3361, 3363, 3365, 3369, 3371, 3372,
3373,
Detection: 3254, 3258, 3 263, 3272, 3277, 3288, 3294, 3296, 3299, 3303, 3317,
3323,
3326, 3329,3336, 3341, 3362, 3366, 3370, 3375,
SEQ ID NO: 16
left Priiner: 3377, 3380, 3383, 3386, 3388,3389, 3390, 3391, 3392, 3395, 3396,
3400, 3401,
rigttt Primer: 3378, 3381, 3384, 3385, 3393, 3397,3399, 3402,
Det.eotion: 3379, 3382, 3387, 3394, 3398,
110
CA 02528853 2005-12-23
SEQ ID NO: 42
left Primer. 3403, 3406, 3408, 3413, 3414, 3417, 3420, 3423, 3428, 3432, 3433,
3434,
3436, 3439, 3440, 3442, 3443, 3445, 3447, 3453, 3455, 3458, 3463, 3466, 3469,
3470, 3471,
3472, 3473, 3474, 3475, 3477, 3478, 3480, 3481, 3483, 3484, 3487, 3490, 3494,
3495, 3496,
3497, 3499, 3500, 3504, 3505, 3508, 3512, 3514, 3515, 3516, 3521, 3525, 3529,
3533, 3534,
3537, 3538, 3540, 3541, 3542, 3543, 3544, 3547, 3548, 3553, 3557, 3558, 3559,
3560, 3562,
3563, 3566, 3571, 3574, 3575, 3577, 3580, 3583, 3584, 3585, 3586, 3592, 3594,
3595, 3597,
3598, 3599, 3601, 3603x 3604, 3607; 3608, 3612, 3619, 3621, 3622, 3626, 3631,
3632, 3637,
3638, 3639, 3640, 3642, 3643, 3644, 3646, 3647, 3649, 3650, 3652, 3656, 3657,
3660, 3663,
3668, 3670, 3673, 3676, 3677, 3678, 3683, 3688, 3690, 3694, 3697, 3700, 3702,
3703, 3704,
3708,
right Primer: 3404, 3409, 3411, 3415, 3418, 3419, 3421, 3424, 3426, 3427,
3429, 3431,
3435, 3437, 3438, 3441, 3444, 3446, 3448, 3450, 3452, 3454, 3456, 3459, 3461,
3462, 3464,
3467, 3476, 3479, 3482, 3485, 3488, 3489, 3491, 3492, 3493, 3501, 3503, 3506,
3509, 3510,
3511, 3513, 3517, 3518, 3519, 3520, 3522, 3524, 3526, 3527, 3528, 3530, 3531,
3535, 3536,
3545, 3549, 3551, 3552, 3554, 3555, 3556, 3561, 3564, 3565, 3568, 3569, 3570,
3573, 3576,
3578, 3581, 3587, 3588, 3589, 3590, 3591, 3593, 3596, 3600, 3602, 3605, 3606,
3609, 3611,
3613, 3614, 3615, 3616, 3617, 3618, 3623, 3624, 3625, 3627, 3629, 3630, 3633,
3634, 3635,
3636, 3641, 3645, 3651, 3653, 3654, 3655, 3658, 3661, 3662, 3665, 3666, 3667,
3669, 3671,
3674, 3679, 3681, 3684, 3686, 3689, 3691, 3692, 3693, 3695, 3696, 3698, 3701,
3705, 3706,
3707, 3709,
Detection: 3405, 3407, 3 410, 3412, 3416, 3422, 3425, 3430, 3449, 3451, 3457,
3460,
3465, 3468, 3486, 3498, 3502, 3507, 3523, 3532, 3539, 3546, 3550, 3567, 3572,
3579, 3582,
3610, 3620, 3628, 3648, 3659, 3664, 3672, 3675, 3680, 3682, 3685, 3687, 3699,
SEQ ID NO: 14
left Primer. 3710, 3714, 3715, 3717, 3718, 3722, 3723, 3729, 3730, 3732, 3735,
3739,
3740, 3741, 3743, 3744, 3745, 3746, 3749, 3753, 3754, 3755, 3756,
right Primer. 3711, 3713, 3719, 3721, 3724, 3725, 3726, 3727, 3728, 3731,
3733, 3736,
111
CA 02528853 2005-12-23
3737, 3738, 3742, 3747, 3748, 3750, 3752, 3757, 3758, 3759, 3760,
Deteation: 3712, 3716, 3720, 3734, 3751,
SEQ ID NO: 43
left Prime.r: 3761, 3764, 3767, 3771, 3773, 3775, 3776, 3777, 3781, 3785,
3786, 3787, 3790,
3791, 3793, 3796, 3797, 3798, 3799, 3805, 3809, 3814, 3817, 3818, 3822, 3823,
3828, 3833,
3836, 3844, 3847, 3850, 3851, 3853, 3856, 3857, 3862, 3873, 3876, 3877, 3880,
3885, 3887,
3889, 3890, 3892, 3893, 3895, 3896, 3899, 3902, 3903, 3905, 3906, 3907, 3908,
3916, 3923,
3926, 3927, 3930, 3935, 3936, 3938, 3939, 3940, 3944, 3945, 3949, 3950, 3951,
3954, 3955,
3957, 3960, 3965, 3970, 3971, 3978, 3979, 3982, 3983, 3984, 3985,
right Primer: 3762, 3765, 3768, 3770, 3772, 3774, 3778, 3779, 3780, 3782,
3784, 3788,
3792, 3794, 3800, 3801, 3802, 3803, 3804, 3806, 3807, 3808, 3810, 3812, 3813,
3815, 3819,
3820, 3821, 3824, 3826, 3827, 3829, 3831, 3832, 3834, 3837, 3838,, 3839, 3840,
3842, 3845,
3848, 3852, 3854, 3858, 3860, 3861, 3863, 3864, 3865, 3866, 3867, 3868, 3869,
3870, 3871,
3872, 3874, 3875, 3878, 3881, 3883, 3884, 3886, 3888, 3891, 3894, 3897, 3898,
3900, 3901,
3904, 3909, 3911, 3914, 3915, 3917, 3919, 3920, 3921, 3922, 3924, 3928, 3931,
3933, 3934,
3937, 3941, 3942, 3943, 3947, 3948, 3952, 3958, 3959, 3961, 3962, 3963, 3964,
3966, 3968,
3972, 3974, 3975, 3976, 3980, 3981, 3986,
Detection: 3763, 3766, 3 769, 3783, 3789, 3795, 3811, 3816, 3825, 3830, 3835,
3841,
3843, 3846, 3849, 3855, 3859, 3879, 3882, 3910, 3912, 3913, 3918, 3925, 3929,
3932, 3946,
3953, 3956, 3967, 3969, 3973, 3977,
SEQ ID NO: 15
left Primer. 3987, 3990, 3993, 3994, 3997, 4000, 4003, 4004, 4006, 4012, 4015,
4017,
4020, 4024, 4027, 4029, 4031, 4032, 4033, 4037, 4038, 4040, 4042, 4047, 4048,
4050, 4051,
4052, 4054, 4055, 4058, 4063, 4066, 4069, 4070, 4072, 4073, 4076, 4078, 4079,
4082, 4083,
4084, 4085, 4086, 4090, 4093, 4094, 4095, 4096, 4099, 4100, 4104, 4106, 4113,
4116, 4119,
4120, 4124, 4127, 4130, 4134, 4135, 4136, 4138, 4142, 4145, 4146, 4149, 4150,
4152, 4153,
4159, 4160, 4161, 4163, 4168, 4169, 4171, 4173, 4176, 4180, 4181, 4185, 4189,
4191, 4192,
112
CA 02528853 2005-12-23
4194, 4197, 4198, 4199, 4201, 4206, 4207, 4208, 4213, 4219, 4220, 4223, 4224,
4225, 4228,
4231, 4232, 4233, 4237, 4238, 4242, 4243, 4246, 4247, 4251, 4254, 4255, 4256,
4258,
right Primer: 3988, 3991, 3995, 3998, 4001, 4005, 4007, 4009, 4010, 4011,
4013, 4016,
4018, 4019, 4021, 4022, 4023, 4025, 4028, 4034, 4035, 4039, 4043, 4045, 4046,
4049, 4056,
4059, 4061, 4064, 4065, 4067, 4068, 4071, 4074, 4075, 4077, 4080, 4087, 4088,
4089, 4092,
4098, 4101, 4102, 4105, 4108, 4109, 4110, 4111, 4112, 4114, 4115, 4117, 4121,
4122, 4123,
4125, 4128, 4129, 4131, 4132, 4133, 4137, 4139, 4141, 4144, 4148, 4151, 4154,
4156, 4157,
4162, 4165, 4166, 4167, 4170, 4172, 4174, 4177, 4179, 4182, 4184, 4186, 4188,
4190, 4195,
4196, 4202, 4204, 4205, 4209, 4211, 4214, 4216, 4217, 4218, 4221, 4226, 4229,
4234, 4236,
4239, 4241, 4244, 4245, 4248, 4249, 4252, 4257, 4259, 4260, 4261,
Datection: 3989, 3992, 3 996, 3999, 4002, 4008, 4014, 4026, 4030, 4036, 4041,
4044,
4053, 4057, 4060, 4062, 4081, 4091, 4097, 4103, 4107, 4118, 4126, 4140, 4143,
4147, 4155,
4158, 4164, 4175, 4178, 4183, 4187, 4193, 4200, 4203, 4210, 4212, 4215, 4222,
4227, 4230,
4235, 4240, 4250, 4253,
SEQ ID NO:4
left Primer: 4262, 4266, 4267, 4268, 4271, 4272, 4275, 4276, 4279, 4281, 4282,
4284,
4285, 4287, 4289, 4290, 4292, 4295, 4297, 4299, 4301, 4306, 4310, 4316, 4317,
4318, 4321,.
4323, 4325, 4328, 4334, 4335, 4337, 4341, 4342, 4343, 4344, 4348, 4350, 4351,
4352, 4354,
4357, 4358, 4359, 4364, 4365, 4366, 4370, 4371, 4372, 4373, 4376, 4378, 4381,
4383, 4384,
4386, 4388, 4396, 4397, 4399, 4400, 4403, 4405, 4407, 4409, 4412, 4415, 4416,
4417, 4424,
4426, 4429, 4433, 4435, 4437, 4442, 4443, 4447, 4450, 4451, 4452, 4455, 4457,
4460, 4461,
4462, 4465, 4466, 4469, 4470, 4474, 4478, 4481, 4484, 4488, 4491, 4494, 4495,
4497, 4498,
4504, 4506, 4509, 4510, 4511, 4514, 4516, 4518, 4520, 4521, 4523, 4525, 4528,
4530, 4533,
4534, 4535, 4538, 4540, 4542, 4545, 4547, 4548, 4553, 4554, 4555, 4556, 4557,
4562, 4564,
4567, 4568, 4569, 4579, 4580, 4582, 4584, 4587, 4589, 4593, 4596, 4597, 4598,
4600, 4601,
4602, 4605, 4606, 4607, 4609, 4610, 4613, 4614, 4616, 4618, 4621, 4624, 4626,
4627, 4628,
4631, 4632, 4636, 4639, 4641, 4644, 4645, 4648,
right Primer: 4263, 4265, 4269, 4270, 4273, 4274, 4277, 4280, 4283, 4286,
4288, 4291,
113
CA 02528853 2005-12-23
4293, 4296, 4298, 4300, 4302, 4303, 4304, 4305, 4307, 4308, 4309, 4311, 4313,
4315, 4319,
4322, 4324, 4326, 4327, 4330, 4331, 4332, 4333, 4336, 4338, 4340, 4345, 4347,
4349, 4353,
4355, 4360, 4361, 4362, 4367, 4368, 4369, 4374, 4375, 4377, 4379, 4382, 4385,
4387, 4389,
4390, 4391, 4392, 4393, 4394, 4395, 4398, 4401, 4406, 4408, 4410, 4411, 4413,
4418, 4420,
4422, 4423, 4425, 4427, 4428, 4430, 4432, 4434, 4436, 4438, 4440, 4441, 4444,
4446, 4448,
4453, 4454, 4456, 4458, 4463, 4464, 4467, 4471, 4472, 4473, 4475, 4476, 4477,
4479, 4480,
4482, 4485, 4486, 4487, 4489, 4490, 4492, 4496, 4499, 4501, 4502, 4503, 4505,
4507, 4508,
4512, 4515, 4517, 4519, 4522, 4524, 4529, 4531, 4536, 4537, 4539, 4541, 4543,
4546, 4549,
4551, 4558, 4559, 4560, 4561, 4563, 4565, 4570, 4571, 4572, 4573, 4574, 4575,
4576, 4578,
4581, 4583, 4585, 4586, 4588, 4590, 4592, 4595, 4599, 4603, 4611, 4612, 4615,
4617, 4619,
4620, 4622, 4630, 4633, 4634, 4635, 4638, 4640, 4642, 4643, 4646, 4647,
Detection: 4264, 4278,4294, 4312, 4314, 4320, 4329, 4339, 4346, 4356, 4363,
4380,
4402, 4404, 4414, 4419, 4421, 4431, 4439, 4445, 4449, 4459, 4468, 4483, 4493,
4500, 4513,
4526, 4527, 4532, 4544, 4550, 4552, 4566, 4577; 4591, 4594, 4604, 4608, 4623,
4625, 4629,
4637,
SEQ ID NO: 7
left Primer: 4649, 4652, 4655, 4659, 4661, 4663, 4664, 4665, 4668, 4671, 4674,
4675,
4676, 4678, 4680, 4683, 4686, 4690, 4696, 4700, 4703, 4704, 4706, 4709, 4711,
4714, 4715,
4717, 4718, 4719, 4720, 4721, 4726, 4727, 4730, 4732, 4738, 4742, 4745, 4749,
4750, 4753,
4755, 4759, 4763, 4768, 4769, 4772, 4773, 4776, 4778, 4779, 4782, 4784, 4789,
4790, 4794,
4797, 4801, 4805, 4807, 4813, 4814, 4819, 4821, 4822, 4827, 4829, 4833, 4834,
4835, 4837,
4838, 4841, 4842, 4843, 4844, 4847, 4849, 4850, 4852, 4853, 4859, 4860, 4861,
4865, 4867,
4868, 4869, 4870, 4873, 4876, 4877, 4878, 4879, 4883, 4885, 4889, 4891, 4892,
4894, 4895,
4897, 4898, 4903, 4906, 4911, 4912, 4918, 4919, 4920, 4924, 4927, 4929, 4932,
4934, 4941,
4942, 4945, 4948, 4949, 4950,
right Primer: 4650, 4653, 4656, 4658, 4660, 4662, 4666, 4667, 4669, 4670,
4672, 4677,
4681, 4684, 4685, 4687, 4688, 4689, 4691, 4693, 4694, 4695, 4697, 4699, 4701,
4702, 4705,
4707, 4708, 4716, 4722, 4724, 4728, 4731, 4733, 4735, 4736, 4737, 4739, 4741,
4744, 4746,
114
CA 02528853 2005-12-23
4748, 4751, 4754, 4756, 4758, 4760, 4761, 4764, 4766, 4767, 4770, 4771, 4774,
4777, 4780,
4783, 4785, 4786, 4787, 4788, 4791, 4792, 4793, 4795, 4798, 4800, 4802, 4804,
4806, 4808,
4810, 4811, 4812, 4815, 4816, 4817, 4818, 4820, 4823, 4824, 4825, 4828, 4830,
4832, 4836,
4839, 4840, 4845, 4846, 4848, 4851, 4854, 4855, 4856, 4857, 4858, 4862, 4864,
4871, 4872,
4874, 4875, 4881, 4882, 4884, 4886, 4887, 4890, 4893, 4896, 4899, 4900, 4901,
4902, 4904,
4905, 4907, 4908, 4909, 4910, 4913, 4915, 4917, 4921, 4922, 4923, 4925, 4926,
4928, 4930,
4935, 4936, 4937, 4938, 4940, 4943, 4944, 4946, 4947, 4951, 4952,
Detection: 4651, 4654, 4 657, 4673, 4679, 4682, 4692, 4698, 4710, 4712, 4713,
4723,
4725, 4729, 4734, 4740, 4743, 4747, 4752, 4757, 4762, 4765, 4775, 4781, 4796,
4799, 4803,
4809, 4826, 4831, 4863, 4866, 4880, 4888, 4914, 4916, 4931, 4933, 4939,
SEQ ID NO: 44
left Primer. 4953,
right Primer: 4954,4956,
Detection: 4955,
SEQ ID NO:1
left Primer. 4957, 4960, 4963, 4967, 4970, 4971, 4972, 4976, 4977, 4985, 4986,
4987,
4989, 4991, 4992, 4997, 4999, 5002, 5003, 5004, 5006, 5010, 5013, 5014, 5015,
5017, 5018,
5019, 5020, 5021, 5024, 5025, 5027, 5031, 5032, 5033, 5037, 5040, 5041, 5042,
5043,
right Primer: 4958, 4961, 4964, 4966, 4968, 4969, 4973, 4974, 4975, 4978,
4979, 4980,
4981, 4982, 4983, 4984, 4988, 4990, 4993, 4995, 4996, 4998, 5000, 5005, 5007,
5009, 5011,
5016, 5022, 5026, 5028, 5030, 5034, 5036, 5038, 5039,
Detection: 4959, 4962, 4965, 4994, 5001, 5008, 5012, 5023, 5029, 5035,
SEQ ID NO:Z
left Primer. 5044, 5047, 5049, 5050, 5052, 5054, 5055, 5057, 5060, 5061, 5064,
right Primer. 5045, 5048, 5051, 5053, 5056, 5058, 5062, 5065,
Detection: 5046, 5059, 5063,
115
CA 02528853 2005-12-23
SEQ ID NO: 45
left Pr6er: 5066, 5069, 5073, 5077, 5080, 5083, 5087, 5093, 5096, 5099, 5100,
5103,
5104, 5106, 5111, 5116, 5119, 5120, 5125, 5128, 5132, 5134, 5135, 5137, 5139,
5140, 5142,
5144, 5148, 5149, 5150, 5155, 5157, 5158, 5160, 5166, 5168, 5170, 5171, 5173,
5176, 5177,
5179, 5184, 5187, 5188, 5190, 5193, 5195, 5198, 5201, 5204, 5208, 5211, 5215,
5216, 5217,
5220, 5221, 5222, 5224, 5226, 5229, 5230, 5231, 5232, 5234, 5237, 5242, 5244,
5245, 5246,
5247, 5249, 5250, 5252, 5254, 5255, 5256, 5261, 5262, 5264, 5265, 5273, 5274,
5276, 5278,
5279, 5281, 5284, 5285, 5286, 5287, 5288, 5289, 5292, 5296, 5300, 5303, 5304,
5307, 5308,
5309, 5313, 5315, 5317, 5318, 5322, 5323, 5326, 5329, 5330, 5333, 5336, 5337,
5341, 5343,
5348, 5349, 5352, 5354, 5355, 5358, 5359, 5361, 5362, 5363, 5364, 5365, 5367,
5369, 5370,
5372, 5375, 5377, 5379, 5382, 5385, 5387, 5393, 5394, 5395, 5398, 5400, 5404,
5407, 5408,
5409, 5412, 5413, 5414, 5416, 5418, 5421, 5424, 5426, 5428, 5431, 5432, 5433,
5434, 5435,
5437, 5440, 5441, 5443, 5448, 5450, 5451, 5454, 5455, 5458, 5460, 5461, 5464,
5468, 5470,
5471, 5474, 5475, 5477, 5482, 5484, 5485, 5487, 5490, 5491, 5492, 5494, 5496,
5498, 5499,
5500, 5501, 5502, 5503, 5504, 5506, 5509, 5510, 5512, 5516, 5517, 5518, 5524,
5525, 5526,
5528, 5531, 5533, 5534, 5535, 5536, 5537, 5540, 5542, 5544, 5545, 5546, 5549,
5550, 5552, .
5553, 5559, 5561, 5566, 5569, 5572, 5573, 5575, 5577, 5578, 5579, 5580, 5581,
5~582, 5588,
5593, 5595, 5596, 5598, 5600, 5601, 5603, 5604, 5605, 5608, 5614, 5615, 5616,
5619, 5622,
5624, 5625, 5628, 5629, 5633, 5634, 5637, 5638, 5639, 5643, 5644, 5645, 5647,
5648, 5649,
5650, 5654, 5662, 5664,
right Primer: 5067, 5070, 5072, 5074, 5075, 5076, 5078, 5081, 5084, 5086,
5088, 5090,
5091, 5094, 5097, 5101, 5105, 5107, 5109, 5110, 5112, 5114, 5115, 5117, 5121,
5123, 5124,
5126, 5129, 5131, 5136, 5143, 5145, 5147, 5151, 5153, 5156, 5159, 5161, 5162,
5163, 5164,
5165, 5167, 5169, 5172, 5174, 5175, 5180, 5181, 5182, 5183, 5185, 5186, 5189,
5191, 5194,
5196, 5199, 5200, 5202, 5203, 5205, 5207, 5209, 5210, 5212, 5214, 5218, 5219,
5223, 5225,
5227, 5233, 5235, 5238, 5239, 5240, 5241, 5248, 5251, 5253, 5257, 5259, 5260,
5263, 5266,
5268, 5269, 5270, 5271, 5272, 5280, 5282, 5290, 5291, 5293, 5295, 5297, 5299,
5301, 5306,
5310, 5311, 5312, 5314, 5316, 5319, 5321, 5324, 5327, 5331, 5332, 5334, 5338,
5339, 5340,
116
CA 02528853 2005-12-23
5342, 5344, 5346, 5347, 5351, 5353, 5356, 5360, 5366, 5368, 5371, 5373, 5376,
5378, 5380,
5383, 5388, 5389, 5391, 5392, 5396, 5399, 5401, 5403, 5405, 5410, 5415, 5417,
5419, 5420,
5422, 5425, 5427, 5429, 5436, 5438, 5442, 5444, 5445, 5446, 5447, 5449, 5452,
5456, 5457,
5459, 5462, 5465, 5466, 5467, 5469, 5472, 5478, 5480, 5481, 5486, 5488, 5493,
5495, 5497,
5505, 5507, 5508, 5511, 5513, 5515, 5519, 5521, 5522, 5527, 5529, 5532, 5538,
5539, 5541,
5543, 5548, 5551, 5554, 5555, 5556, 5557, 5558, 5560, 5562, 5563, 5564, 5565,
5567, 5568,.
5570, 5571, 5574, 5576, 5583, 558S, 5586, 5587, 5589, 5591, 5592, 5594, 5597,
5599, 5602,
5606, 5609, 5611, 5612, 5618, 5620, 5623, 5626, 5627, 5630, 5632, 5635, 5640,
5642, 5646,
5651, 5652, 5656, 5657, 5659, 5660, 5661, 5663, 5665,
Detection: 5068, 5071, 5 079, 5082, 5085, 5089, 5092, 5095, 5098, 5102, 5108,
5113,
5118, 5122, 5127, 5130, 5133, 5138, S141, 5146, 5152, 5154, 5178, 5192, 5197,
5206, 5213,
5228, 5236, 5243, 5258, 5267, 5275, 5277, 5283, 5294, 5298, 5302, 5305, 5320,
5325, 5328,
5335, 5345, 5350, 5357, 5374, 5381, 5384, 5386, 5390, 5397, 5402, 5406, 5411,
5423, 5430,
5439, 5453, 5463, 5473, 5476, 5479, 5483, 5489, 5514, 5520, 5523, 5530, 5547,
5584, 5590,
5607, 5610, 5613, 5617, 5621, 5631, 5636, 5641, 5653, 5655, 5658, 5666,
SEQIDNO:9
left Fruner: 5667, 5670, 5672, 5673, 5676, 5678, 5679, 5681, 5682, 5683, 5686,
5687;
5688, 5690, 5694, 5697, 5698, 5699, 5700, 5701, 5704, 5708, 5709, 5711, 5713,
5714, 5717,
5721, 5723, 5724, 5727, 5728, 5733, 5734, 5735, 5736, 5739, 5740, 5741, 5742,
5743,
right Primer. 5668, 5671, 5674, 5677, 5680, 5684, 5689, 5691, 5693, 5696,
5702, 5705,
5707, 5710, 5712, 5715, 5718, 5720, 5722, 5725, 5729, 5730, 5731, 5732, 5737,
Detection: 5669, 5675, 5685, 5692, 5695, 5703, 5706, 5716, 5719, 5726, 5738,
SEQ ID NO: 46
left Primer: 5744, 5747, 5750, 5753, 5759, 5762, 5763, 5770, 5775, 5777, 5778,
5780,
5782, 5785, 5787, 5791, 5792, 5793, 5794, 5797, 5801, 5803, 5808, 5810, 5812,
5814, 5816,
5817, 5824, 5832, 5833, 5834, 5835, 5838, 5840, 5841, 5844, 5849, 5853, 5854,
5857, 5859,
5860, 5862, 5863, 5865, 5868, 5874, 5882, 5883, 5885, 5887, 5894, 5895, 5896,
5897, 5902,
117
CA 02528853 2005-12-23
5904, 5906, 5909, 5912, 5915, 5918, 5919, 5920, 5922, 5923, 5926, 5927, 5928,
5930, 5932,
5933, 5934, 5935, 5939, 5940, 5946, 5947, 5948, 5949, 5952, 5954, 5955, 5956,
5958, 5959,
5961, 5962, 5964, 5965, 5968, 5970, 5971, 5972, 5973, 5977, 5978, 5980, 5983,
5986, 5987,
5992, 5993, 5996, 5997, 6000, 6002, 6003, 6004, 6005, 6006, 6008, 6009, 6010,
6011, 6012,
6013, 6017, 6018, 6019, 6020, 6025, 6027, 6030, 6033, 6035, 6036, 6039, 6041,
6042, 6045,
6049, 6050, 6051, 6055, 6059, 6063, 6064, 6065, 6067, 6070, 6072, 6073, 6075,
6077, 6078,
6081,
right Primer: 5745, 5748, 5751, 5754, 5756, 5758, 5760, 5764, 5766, 5767,
5768, 5769,
5771, 5773, 5774, 5776, 5779, 5781, 5783, 5786, 5788, 5789, 5790, 5795, 5796,
5798, 5800,
5802, 5804, 5806, 5807, 5809, 5811, 5813, 5818, 5820, 5821, 5822, 5823, 5825,
5826, 5827,
5828, 5830, 5831, 5836, 5837, 5839, 5842, 5843, 5845, 5846, 5847, 5848, 5850,
5852, 5855,
5856, 5858, 5861, 5864, 5866, 5867, 5870, 5871, 5872, 5873, 5875, 5876, 5877,
5878,5879,
5880, 5881, 5884, 5886, 5888, 5889, 5890, 5891, 5892, 5893, 5898, 5899, 5900,
5903, 5905,
5907, 5910, 5913, 5916, 5921, 5924, 5929, 5931, 5936, 5937, 5938, 5942, 5943,
5944, 5945,
5950, 5953, 5957, 5960, 5963, 5966, 5969, 5975, 5976, 5981, 5984, 5988, 5990,
5991, 5994,
5998, 5999, 6001, 6007, 6014, 6015, 6016, 6021, 6023, 6024, 6026, 6028, 6031,
6032, 6034,
6037, 6038, 6040, 6043, 6046, 6048, 6053, 6054, 6056, 6058, 6060, 6062, 6066,
6068, 6074,
6076, 6079, 6083, 6084,
Detection: 5746, 5749,5 752, 5755, 5757, 5761, 5765, 5772, 5784, 5799, 5805,
5815,
5819, 5829, 5851, 5869, 5901, 5908, 5911, 5914, 5917, 5925, 5941, 5951, 5967,
5974, 5979,
5982, 5985, 5989, 5995, 6022, 6029, 6044, 6047, 6052, 6057, 6061, 6069, 6071,
6080, 6082,
SEQ ID NO: 10
left Psimer. 6085, 6089, 6090, 6093, 6096, 6098, 6100, 6106, 6110, 6111, 6114,
6118,
6121, 6127, 6130, 6133, 6135, 6137, 6138, 6141, 6143, 6145, 6148, 6150, 6151,
6152, 6153,
6154, 6156, 6159, 6160, 6162, 6163, 6166, 6167, 6168, 6169, 6170, 6171, 6172,
6175, 6177,
6180, 6183, 6187, 6188, 6190, 6193, 6194, 6195, 6196, 6198, 6201, 6202, 6204,
6207, 6211,
6212, 6214, 6215, 6216, 6218, 6219, 6220, 6222, 6225, 6226, 6230, 6235, 6239,
6241, 6242,
6243, 6244, 6245, 6247, 6250, 6252, 6254, 6259, 6260, 6261, 6264, 6265, 6268,
6270, 6273,
118
CA 02528853 2005-12-23
6275, 6278, 6280, 6282, 6286, 6287, 6288, 6291, 6293, 6298, 6299, 6300, 6302,
6303, 6305,
6307, 6308, 6312, 6313, 6314, 6315, 6316, 6317, 6318, 6320, 6321, 6323, 6325,
6327, 6328,
6334, 6335, 6337, 6340, 6341, 6344, 6348, 6350, 6353, 6354, 6357, 6360, 6362,
6364, 6368,
6372, 6374, 6377, 6378, 6383, 6386, 6392, 6394, 6398, 6400, 6404, 6405, 6409,
6410, 6411,
6417, 6418, 6424, 6426, 6429, 6431, 6432, 6433, 6435, 6436, 6437, 6438, 6440,
6442, 6445,
6446, 6448, 6449, 6452, 6455, 6457, 6458, 6461, 6462, 6463, 6464, 6465, 6470,
6473, 6475,
6478, 6480, 6481, 6484, 6485, 6486, 6489, 6491, 6492, 6494, 6498, 6502, 6504,
6505, 6507,
6511, 6513, 6514,
right Primer: 6086, 6088, 6091, 6094, 6097, 6099, 6101, 6103, 6105, 6108,
6112, 6115,
6116, 6117, 6119, 6120, 6122, 6123, 6125, 6126, 6128, 6131, 6132, 6134, 6136,
6139, 6142,
6144, 6146, 6149, 6155, 6157, 6161, 6164, 6173, 6174, 6176, 6178, 6181, 6182,
6184, 6186,
6189, 6191, 6192, 6197, 6199, 6200, 6205, 6206, 6208, 6210, 6213, 6221, 6223,
6224, 6227,
6229, 6231, 6232, 6233, 6234, 6236, 6237, 6238, 6240, 6246, 6248, 6251, 6253,
6255, 6256,
6257, 6258, 6262, 6266, 6269, 6271, 6272, 6274, 6276, 6277, 6281, 6283, 6284,
6285, 6289,
6292, 6294, 6295, 6296, 6297, 6301, 6304, 6306, 6309, 6310, 6311, 6319, 6322,
6324, 6326;
6329, 6330, 6331, 6333, 6336, 6338, 6342, 6345, 6347, 6349, 6351, 6355, 6356,
6358, 6359,
6361, 6365, 6367, 6369, 6371, 6373, 6375, 6376, 6379, 6381, 6382, 6384, 6387,
6388, 6389,
6390, 6393, 6395, 6397, 6399, 6401, 6402, 6403, 6406, 6407, 6412, 6413, 6414,
6415, 6416,
6419, 6420, 6421, 6422, 6423, 6425, 6427, 6428, 6434, 6441, 6443, 6444, 6447,
6450, 6451,
6453, 6459, 6460, 6466, 6467, 6468, 6469, 6471, 6472, 6474, 6476, 6479, 6482,
6487, 6490,
6493, 6496, 6497, 6499, 6500, 6501, 6503, 6506, 6508, 6510, 6512,
Deteotioa: 6087, 6092, 6 095, 6102, 6104, 6107, 6109, 6113, 6124, 6129, 6140,
6147,
6158, 6165, 6179, 6185, 6203, 6209, 6217, 6228, 6249, 6263, 6267, 6279, 6290,
6332, 6339,
6343, 6346, 6352, 6363, 6366, 6370, 6380, 6385, 6391, 6396, 6408, 6430, 6439,
6454, 6456,
6477, 6483, 6488, 6495, 6509,
SEQ ID NO: 11
left Primer. 6515, 6518, 6521, 6522, 6523, 6524, 6528, 6532, 6535, 6539, 6549,
6560,
6561, 6564, 6566, 6567, 6570, 6573, 6576, 6577, 6578, 6579, 6583, 6584, 6585,
6586, 6587,
119
CA 02528853 2005-12-23
6590, 6591, 6594, 6596, 6597, 6603, 6605, 6608, 6609, 6610, 6611, 6612, 6615,
6625, 6630,
6632, 6633, 6634, 6638, 6639, 6642, 6643, 6644, 6647, 6650, 6655, 6657, 6658,
6659, 6661,
6663, 6664, 6665, 6669, 6671, 6674, 6677, 6678, 6679, 6680, 6681, 6682, 6686,
6687, 6688,
6689, 6690, 6691, 6693, 6695, 6696, 6697, 6699, 6701, 6703, 6704, 6705, 6706,
6707, 6708,
6710, 6711, 6712, 6713, 6717, 6718, 6720, 6721, 6722, 6725, 6727, 6732, 6734,
6735, 6739,
6741, 6745, 6748, 6751, 6755, 6759, 6762, 6764, 6767, 6770, 6772, 6773, 6774,
6777, 6778,
6782, 6785, 6786, 6787, 6790, 6791, 6794, 6795, 6796, 6798, 6800, 6802, 6803,
6806, 6811,
6814, 6815, 6818, 6824, 6829, 6834, 6835, 6842,
right Primer: 6516, 6519, 6525, 6526, 6527, 6529, 6531, 6533, 6536, 6537,
6538, 6540,
6541, 6542, 6543, 6544, 6545, 6546, 6547, 6548, 6550, 6551, 6552, 6553, 6554,
6556, 6558,
6562, 6563, 6565, 6568, 6571, 6572, 6575, 6580, 6582, 6588, 6589, 6592, 6595,
6598, 6599,
6600, 6601, 6602, 6604, 6606, 6607, 6613, 6614, 6617, 6618, 6619, 6620, 6621,
6623, 6624,
6627, 6628, 6629, 6631, 6635, 6640, 6641, 6645, 6646, 6649, 6651, 6652, 6653,
6656, 6660,
6662, 6666, 6668, 6670, 6672, 6675, 6676, 6683, 6685, 6692, 6694, 6698, 6700,
6702, 6709,
6714, 6715, 6716, 6719, 6723, 6726, 6728, 6730, 6731, 6736, 6738, 6740, 6742,
6744, 6746,
6747, 6749, 6752, 6754, 6756, 6757, 6758, 6760, 6761, 6765, 6769, 6775, 6779,
6781, 6783,
6784, 6789, 6792, 6797, 6799, 6801, 6804, 6805, 6807,,6809, 6810, 6812, 6816,
6817, 6819,
6821, 6822, 6825, 6827, 6828, 6830, 6831, 6832, 6833, 6836, 6837, 6838, 6839,
6840, 6841,
6843,
Dctection: 6517, 6520, 6 530, 6534, 6555, 6557, 6559, 6569, 6574, 6581, 6593,
6616,
6622, 6626, 6636, 6637, 6648, 6654, 6667, 6673, 6684, 6724, 6729, 6733, 6737,
6743, 6750,
6753, 6763, 6766, 6768, 6771, 6776, 6780, 6788, 6793, 6808, 6813, 6820, 6823,
6826,
SEQ ID NO: 12
left Primea: 6844, 6848, 6850, 6852, 6854, 6857, 6859, 6861, 6862, 6865, 6868,
6870,
6871, 6872, 6874, 6875, 6876, 6880, 6883, 6886, 6887, 6888, 6890, 6891, 6894,
6896, 6897,
6898, 6899, 6900, 6901, 6902, 6904, 6905, 6906, 6910, 6912, 6917, 6920,
right Primer: 6845, 6847, 6849, 6851, 6853, 6855, 6858, 6860, 6863, 6866,
6869, 6873,
6877, 6879, 6881, 6882, 6884, 6889, 6892, 6893, 6895, 6907, 6908, 6909, 6911,
6913, 6915,
120
IZI
'Z1IL 'IIIL '80IL 'LOIL '90IL '501L '~OIL 'I0I1. '860L '560L 'b60L '~60L 'Z60L
'~80L 'Z80L
'ISOL '080L 'BLOL 'SLOL '~L0L 'ILOL 'OLOL '890L '990L 'S90L '~901. 'Z90L 'I90L
'LSOL 'VSOL
'~S0L 'I S0L '6irOL 'BbOL 'LtOL 'ML 'SbOL 'bbOL '~bOL 'ZtrOL 'Ii-OL '6~0L
'8~0L 'S~OL 't-~OL
'~~OL 'I~OL '6Z0L 'BZOL '9ZOL 'SZOL '~ZOL 'OZOL '8I0L '910L 'bIOL 'ZIOL 'IIOL
'0I0L '600L
'L00L 'SOOL '~OOL '100L '6669 '9669 '~669 '1669 '0669 '6869 'L869 'b869 ugum.d
ASu SZ
'OZtrL 'LIt-L '9I$,L 'SIbL '~IVL '0Ii-L 'SOtiL 'ZOtiL 'IOtiL 'L6~L 'b6~L 'Z6~L
'i6~L
'06~L 'L8~L 'S8~L 'I8~L '8L~L 'SL~L 'bL~L 'ZL~L 'Z9~L '09~L '8S~L 'SS~L '~S~L
'09~L 'tib~L
'It~L 'Ob~L '6~~L '9~~L 'b~~L 'Z~~L 'I~~L '6Z~L 'SZ~L 'bZ~L 'IZ~L 'OZ~L 'SI~L
'bI~L 'II~L
'OI~L '60~L '90~L '~0~L 'ZO~L '66ZL 'S6ZL 'I6ZL 'L8ZL 'ti8ZL '~8ZL 'I8ZL '08ZL
'6LZL '8LZL
',GLZ,L '9LZL 'ZLZL '69ZL '89ZL '99ZL 'S9ZL 'tr9ZL '~9ZL '09ZL '95ZL 'SSZL
'ISZL '6tiZL '8frZL OZ
'9VZL 'SbZL '~trZL '6~ZL 'L~ZL 'S~ZL '~~ZL '0~ZL 'BZZL 'LZZL 'IZZL 'OZZL '91ZL
'tiIZL '~IZL
'80ZL''L40ZL 'SOZL 'bOZL '~OZL 'ZOZL '661L '1-6IL 'Z6IL 'L8IL '98tL '581L
'EBIL '181L '081L
'8L1L 'SLIL 'ZLIL '89IL '99IL 'S91L '~9IL '091L '6SIL 'BSIL '6fiiL 'BVIL '9bIL
'trblL 'ObIL
'6~IL '8~IL '9~IL '5~1L '~~IL 'I~IL 'hZIL '~ZIL 'IZIL '811L 'SIIL 'VIIL '~IIL
'OIIL '60IL
'ZOIL '001L '460L '960L 'I60L '060L '880L '880L 'LSOL '980L 'S80L '480L 'LLOL
'9LOL 'bLOL SI
'ZLOL 'L90L 'tr90L '090L '6S0L '850L '9S0L 'SSOL 'ZSOL 'OSOL 'OvOL 'L~OL '9~0L
'Z~OL '0~OL
'LZOL 11ZOL 'ZZOL 'L10L '~I0L 1900L 'ZOOL '8669 'S669 'Z669 '9869 '~869 ~ZmPd
NaI
vox cii aas
'Z869 48L69 4Ib69 '8~69 '9Z69 '~Z69 -U?PQlaQ 01
'I869 'LL69 'SL69 'bL69 'iL69 '0L69 'L969 '9969 '~969 'Z969 '0969 '8569 '1569
'9569 'ZS69
'0569 'SV69 '"69 'Zb69 '9~69 'b~69 'Z~69 'I~69 '0~69 '6Z69 'SZ69 'ZZ69 :-Mm.ud
lqfP
'0869 '6L69
'91.69 '~1.69 'ZL69 '6969 '8969 '9969 '17969 '1969 46569 '9969 '17569 '~569
'1S69 '61,69 *69
'Ltr69 '91r69 '~1169 '0tr69 '6~69 'L~69 'S~69 '~~69 '8Z69 'LZ69 'tiZ69 'IZ69
=Mud $ai S
~I :ON (IIbas
'6169 'tii69 '~069 '5889 '8L89 'L989 't989 '9589 '9V89 =uo9mpQ
'8169 '9I69
~Z-ZT-SOOZ ~588ZSZ0 Fi'J
CA 02528853 2005-12-23
7116, 7119, 7122, 7125, 7126, 7127, 7128, 7129, 7130, 7132, 7134, 7137, 7141,
7142, 7143,
7147, 7150, 7152, 7154, 7155, 7156, 7157, 7161, 7167, 7170, 7171, 7173, 7174,
7176, 7177,
7179, 7182, 7184, 7188, 7189, 7190, 7191, 7193, 7195, 7196, 7197, 7198, 7200,
7206, 7210,
7211, 7212, 7215, 7217, 7218, 7219, 7222, 7223, 7224, 7225, 7226, 7229, 7231,
7232, 7236,
7240, 7241, 7242, 7244, 7247, 7250, 7253, 7254, 7257, 7258, 7259, 7261, 7262,
7267, 7270,
7271, 7273, 7274, 7275, 7282, 7285, 7289, 7290, 7292, 7293, 7294, 7296, 7297,
7298, 7301,
7304, 7307, 7308, 7312, 7316, 7317, 7318, 7319, 7322, 7326, 7328, 7330, 7333,
7335, 7337,
7342, 7345, 7347, 7348, 7349, 7351, 7354, 7356, 7357, 7359, 7361, 7363, 7364,
7365, 7366,
7367, 7368, 7369, 7370, 7371, 7373, 7376, 7379, 7382, 7383, 7384, 7386, 7388,
7389, 7393,
7395,7398,7400,7403,7404,7407,7408,7409,7411,7419,7421,
Detection: 6985, 6988, 6994, 6997, 7000, 7004, 7008, 7015, 7019, 7021, 7069,
7079,
7099, 7104, 7117, 7120, 7145, 7151, 7153, 7162, 7164, 7169, 7201, 7209, 7234,
7238, 7252,
7286, 7288, 7300, 7305, 7313, 7323, 7327, 7338, 7343, 7346, 7352, 7377, 7380,
7396, 7399,
7406, 7412, 7414, 7418,
SEQ II) NO: 47
left Prnner: 7422, 7425, 7427, 7435, 7438, 7439, 7440, 7442, 7444, 7448, 7449,
7456,
1 7459, 7462, 7463, 7465, 7466, 7467, 7470, 7471, 7474, 7476, 7477, 7480,
7481, 7485, 7486,
7489, 7490, 7493, 7496, 7500, 7503, 7508, 7512, 7518, 7519, 7520, 7522, 7524,
7525, 7527,
7528, 7529, 7532, 7540, 7542, 7543, 7545, 7549, 7552, 7554,
right Primer: 7423, 7426, 7428, 7430, 7431, 7432, 7433, 7434, 7436, 7445,
7446, 7447,
7450, 7451, 7452, 7453, 7454, 7455, 7457, 7460, 7464, 7468, 7472, 7475, 7478,
7479, 7482,
7483, 7484, 7487, 7488, 7492, 7494, 7495, 7497, 7498, 7499, 7501, 7505, 7506,
7507, 7509,
7510, 7511, 7513, 7515, 7516, 7517, 7521, 7523, 7526, 7530, 7533, 7534, 7535,
7536, 7537,
7538, 7539, 7541, 7544, 7546, 7547, 7548, 7550,
Detection: 7424, 7429,7437, 7441, 7443, 7458, 7461, 7469, 7473, 7491, 7502,
7504,
7514, 7531, 7551, 7553,
SEQ ID NO: 18
122
~ZI
'ZLBL 'ILBL '0L8L '698L '998L 't98L 'I98L '8S8L '998L '558L 't'S8L '~S8L '08L
'8v8L 'LbBL
'9"L 'bb8L 'ZV8L 'Ifi8L '0"L '8~8L '9~8L '9~84 '~~8L '0~8L '6Z8L 'LZBL 'SZSL
'IZ8L '8I8L
'9I8L 11I8L '~I8L 'ZI8L '808L '~08L 'Z08L '008L '86LL '96LL 'mLL ULL ULL 'I6LL
'68LL
'SBLL 'f~8LL '~8LL 'I8LL '6LLL '9LLL '~LLL 'ILLL '89LL 'L9LL '99LL 'Z9LL
301M.d Tou
'I66L '886L SZ
'L86L '986L 'S86L '~86L 'Z86L '186L '086L '6L6L '9L6L '~L6L 'ZL6L 'IL6L '896L
'b96L '~96L
'LS6L '996L '5S6L '1pS6L '~S6L 'ZS6L 'I96L '0S6L '81,6L '9b6L 'Sti6L 'Ib6L
'L~6L 'tr~6L '~~6L
'8Z6L '9Z6L 'SZ6L 'ZZ6L 'LI6L '~I6L '0I6L '606L 'LO6L 'b06G 'ZO6L '006L '968L
'b68L 'Z68L
'688L 'S88L 'I88L 'VL8L '898L 'S98L '~98L '098L 'LS8L 'Z58L 'I58L '0181 '~b8L
'6~8L 'L~8L
'b~8L 'Z~8L '8Z8L 'tiZ8L '~Z8L 'ZZ8L 'OZ8L '6I8L 'LI8L '9I8L 'II8L '0I8L '608L
'L08L 'S08L OZ
'66LL ULL ULL '88LL '98LL 'Z8LL 'BLLL 'SLLL 'YLLL 'OLLL 'b9LL 'I9LL ~~T~d PI
oz :ou cu ass
'90LL '969L '~69L '899L 'S99L ' ~59L 'ML 'i'~9L
'Z~9L '0~9L 'SZ9L 'ZI9L 'S09L 'Z09L 'Z65L 'ZLSL '69SL 'V9S L'Z9SL 'LSSL
MoW1a1a(i SI
'09LL '6SLL
'LSLL 'St~LL '~bLL 'Zf~LL '6~LL 'L~LL 'Z~LL ' I ~LL 'OELL 'LZLL 'SLLL 'VZLL
'~ZLL 'ZZLL 'IZLL
'6ILL 'LILL 'SILL 'OILL '80LL '50LL '~OLL 'OOLL '869L '169L '569L 'Z69L '8891.
'9891. 'mL
'I89L '089L '6L9L '8L9L 'SL9L '~L9L ' I L9L 'OL9L '699L '09L '999L 'E99L 'Z99L
'099L 46S9L
'8S9L 'L59L '9S9L *S9L 'Z59L '099L '09L '9V9L 'S'b9L 'iV9L 'Zb9L 'It9L '0V9L
'6~9L '8~9L 01
'L~9L 'I ~9L '8Z9L '9Z9L 'tiZ9L 'ZZ9L '6I9L 'LI9L '919L 'irI9t '~I9L 'I I9L
'809L 'L09L 't-09L
'I09L '66SL 'S6SL 'b65L '~6SL 'I69L 'i-85L '~8SL '895L '~9SL '19SL '9SSL =M!d
Wu
'89LL '9SLL 'SSLL *SLL '~SLL 'ZSLL ' I SLL 'OSLL '6ti.LL 'WL
'L'PLL '9'bLL '6VLL 'IftL '011 ULL '9~LL 'S~LL 'ti~LL '~~LL '6ZL4 '8ZLL '9ZLL
'OZLL 'BILL
'9ILL 'tiILL '~ILL 'ZILL 'IILL '60LL 'LOLL '1i0LL 'ZOLL 'IOLL '669L 'fi69L
'169L '069L '689L S
'L89L 'S89L '~891.'Z89L 'LL9L '9L9L 'tL9L 'ZL9L '999L 'I99L 'SS9L 'I59L 'Ltr9L
'~tr9L '9~9L
'5~9L '~~9L '6Z9L 'LZ9L '~Z9L 'IZ9L '0Z9L '8I9L 'SI9L '0I9L '609L '909L '~09L
'009L '86SL
'L6SL '969L '069L '68SL '885L 'L85L '98SL 'S8SL 'ZBSL 'IBSL '08SL '6LSL 'BLSL
'LLSL '9LSL
'SLSL 'tLSL '~LSL 'ILSL 'OLSL 'L95L '995L 'S9SL '09SL '659L 'BSSL 'SSSL uOtWd
)PI
~Z-ZT-SOOZ ~588ZSZ0 Fi'J
CA 02528853 2005-12-23
7873, 7875, 7877, 7878, 7879, 7880, 7882, 7884, 7886, 7888, 7890, 7897, 7899,
7903, 7905,
7912, 7914, 7916, 7918, 7919, 7920, 7921, 7923, 7929, 7931, 7932, 7936, 7938,
7939, 7940,
7942, 7943, 7944, 7947, 7949, 7958, 7960, 7962, 7965, 7967, 7969, 7970, 7974,
7977, 7989,
7990,
S Detection: 7763, 7766,7769, 7772, 7777, 7780, 7787, 7790, 7801, 7804, 7806,
7826,
7831, 7850, 7859, 7862, 7867, 7876, 7883, 7887, 7891, 7893, 7895, 7898, 7901,
7906, 7908,
7911, 7915, 7924, 7927, 7930, 7935, 7959, 7961, 7966, 7975, 7978, 7984,
SEQ ID NO: 48
left Primer: 7992, 7995, 7996, 8000, 8003, 8006, 8007, 8010, 8012, 8015, 8016,
8018,
8019, 8026, 8027, 8030, 8034, 8035, 8038, 8039, 8043, 8044, 8046, 8049, 8050,
8052, 8053,
8055, 8056, 8062, 8064, 8066, 8067, 8068, 8069, 8070, 8071, 8074, 8076, 8077,
8078, 8079,
8080, 8081, -
right Primer: 7993, 7997, 7999, 8001, 8004, 8005, 8008, 8009, 8011, 8013,
8017, 8020,
8021, 8022, 8023, 8024, 8025, 8028, 8031, 8033, 8036, 8040, 8042, 8047, 8051,
8054, 8057,
8059, 8060, 8061, 8065, 8072, 8073, 8075, 8082, 8083, 8084, 8085, 8087, 8088,
8089
Detection: 7994, 7998, 8002, 8014, 8029, 8032, 8037, 8041, 8045, 8048, 8058,
8063,
8086,
SEQ ID NO: 50
left Primer 8090, 8093, 8095, 8098, 8100, 8101, 8102, 8104, 8107, 8110, 8111,
8114,
8115, 8116, 8118, 8122, 8124, 8126, 8127, 8129, 8132, 8134, 8136, 8138, 8142,
8143, 8144,
8146, 8151, 8157,. 8160, 8163, 8165, 8168, 8169, 8170, 8173, 8174, 8175, 8176,
8178, 8181,
8185, 8187, 8188, 8189, 8190, 8191, 8193, 8194, 8200, 8203, 8206, 8208, 8209,
8212, 8214,
8216, 8217, 8220, 8222, 8224, 8228, 8230,
right Primer: 8091, 8094, 8096, 8099, 8103, 8105, 8108, 8112, 8113, 8117,
8119, 8121,
8123, 8125, 8128, 8130, 8131, 8133, 8135, 8137, 8139, 8145, 8147, 8148, 8149,
8150, 8152,
8153, 8154, 8155, 8156, 8158, 8161, 8164, 8166, 8167, 8171, 8177, 8179, 8180,
8182, 8183,
8184, 8186, 8192, 8195, 8197, 8198, 8199, 8201, 8202, 8204, 8207, 8210, 8213,
8218, 8221,
124
CA 02528853 2005-12-23
8223, 8225, 8227, 8229, 8231,
Detectiog: 8092, 8097, 8106, 8109, 8120, 8140, 8141, 8159, 8162, 8172, 8196,
8205,
8211, 8215, 8219, 8226,
SEQ ID NO:19
left Primer. 8232, 8235, 8236, 8237, 8238, 8244, 8245, 8247, 8250, 8251, 8253,
8254,
8255, 8256, 8258, 8259, 8260, 8263, 8267, 8269, 8270, 8273, 8274, 8277, 8280,
8281, 8289,
8292, 8293, 8294, 8295, 8296, 8297, 8298, 8301, 8305, 8306, 8310, 8311, 8312,
8315, 8317,
8318, 8322, 8323, 8324, 8327, 8328, 8329, 8330, 8333, 8336, 8339, 8340, 8347,
8349, 8350,
8352, 8353, 8356, 8359, 8360, 8366, 8369, 8370, 8372, 8374, 8376, 8377, 8379,
8381, 8382,
8383,
right Primer. 8233, 8239, 8240, 8242, 8243, 8246, 8248, 8252, 8257, 8261,
8264, 8266,
8268, 8271, 8275, 8278, 8282, 8284, 8285, 8286, 8287, 8288, 8291, 8299, 8302,
8303, 8304,
8307, 8309, 8313, 8314, 8316, 8319, 8321, 8325, 8326, 8331, 8332, 8334, 8335,
8337, 8341,
8342, 8343, 8344, 8345, 8346, 8348, 8351, 8354, 8357, 8361, 8362, 8363, 8364;
8365, 8367,
8371, 8373, 8375, 8378,
Detection: 8234, 8241, 8 249, 8262, 8265, 8272, 8276, 8279, 8283, 8290, 8300,
8308,
8320, 8338, 8355, 8358, 8368, 8380,
SEQ ID NO:17
left .Primer. 8384, 8387, 8390, 8391, 8397, 8398, 8401, 8402, 8405, 8406,
8409, 8414,
8418, 8419, 8422, 8425, 8428, 8431, 8432, 8433, 8435, 8437, 8440, 8444, 8449,
8452, 8454,
8455, 8457, 8462, 8464, 8466, 8468, 8469, 8472, 8475, 8476, 8478, 8479, 8481,
8484, 8487,
8488, 8490, 8493, 8496, 8497, 8498, 8501, 8505, 8508, 8511, 8513, 8515, 8517,
8518, 8519,
8521, 8522, 8523, 8526, 8528, 8530, 8531, 8532, 8534, 8536, 8541, 8544, 8547,
8551, 8554,
8556, 8558, 8560, 8562, 8563, 8566, 8567, 8568, 8570, 8571, 8574, 8575, 8576,
8577, 8580,
8581, 8585, 8588, 8591, 8594, 8601, 8602, 8603, 8608, 8609, 8613, 8614, 8617,
8618, 8619,
8620, 8623, 8624, 8628, 8629, 8630, 8633, 8634, 8635, 8636, 8638, 8643, 8645,
8648, 8654,
8657, 8658, 8659, 8660, 8661, 8662, 8666, 8670, 8672, 8674, 8676, 8681, 8683,
8686, 8688,
125
CA 02528853 2005-12-23
8689, 8691, 8692, 8693, 8695, 8696, 8700, 8701, 8702, 8703, 8704, 8705, 8709,
8716, 8717,
8719, 8722, 8725, 8727, 8728, 8729, 8732, 8733, 8734, 8735, 8736,
right Primer. 8385, 8388, 8392, 8394, 8395, 8399, 8403, 8407, 8410, 8411,
8412, 8413,
8415, 8416, 8420, 8423, 8424, 8426, 8429, 8434, 8436, 8438, 8442, 8445, 8446,
8447, 8448,
8450, 8451, 8456, 8458, 8460, 8461, 8463, 8465, 8467, 8470, 8473, 8474, 8477,
8480, 8482,
8485, 8486, 8489, 8491, 8492, 8494, 8499, 8500, 8502, 8503, 8504, 8506, 8510,
8512, 8514,
8516, 8520, 8524, 8527, 8529, 8533, 8535, 8537, 8539, 8542, 8545, 8546, 8548,
8550, 8552,
8555, 8557, 8564, 8565, 8569, 8572, 8578, 8582, 8583, 8586, 8587, 8589, 8592,
8595, 8596,
8597, 8598, 8599, 8600, 8604, 8606, 8607, 8610, 8612, 8615, 8616, 8621, 8625,
8627, 8631,
8632, 8637, 8639, 8640, 8641, 8642, 8644, 8646, 8647, 8649, 8650, 8651, 8652,
8653, 8655,
8663, 8665, 8667, 8668, 8669, 8671, 8673, 8675, 8677, 8678, 8679, 8680, 8682,
8684, 8685,
8687, 8690, 8694, 8697, 8698, 8699, 8706, 8707, 8708, 8710, 8711, 8712, 8713,
8714, 8715,
8718, 8720, 8723, 8726, 8730, 8737,
Detection: 8386, 8389, 8393, 8396, 8400, 8404, 8408, 8417, 8421, 8427, 8430,
8439,
8441, 8443, 8453, 8459, 8471, 8483, 8495, 8507, 8509, 8525, 8538, 8540, 8543,
8549; 8553,
8559, 8561, 8573, 8579, 8584, 8590, 8593, 8605, 8611, 8622, 8626, 8656, 8664,
8721, 8724,
8731,
SEQ ID NO: 23
left Primer: 8738, 8741, 8744, 8747, 8750, 8751, 8753, 8754, 8755, 8756, 8759,
8760,
8762, 8763, 8766, 8767, 8768, 8769, 8770, 8772, 8775, 8776, 8777, 8779, 8782,
8783, 8784,
8785, 8786, 8787, 8788, 8789, 8790, 8793, 8794, 8796, 8798, 8799, 8801, 8803,
8804, 8806,
8807, 8809, 8812, 8813, 8815, 8820, 8823, 8826, 8829, 8830, 8832, 8835, 8836,
8837; 8842,
8845, 8846, 8847, 8851, 8853, 8856, 8857, 8859, 8861, 8866, 8739, 8878, 8879,
8880, 8884,
8891, 8893, 8895, 8898, 8900, 8901, 8909, 8912,
right Primer: 8739, 8742, 8745, 8748, 8749, 8752, 8757, 8758, 8761, 8764,
8771, 8773,
8778, 8780, 8791, 8797, 8800, 8802, 8805, 8808, 8810, 8814, 8816, 8818, 8819,
8821, 8824,
8827, 8828, 8831, 8833, 8838, 8840, 8841, 8755, 8843, 8844, 8848, 8850, 8852,
8854, 8855,
8863, 8864, 8865, 8867, 8868, 8869, 8870, 8871, 8872, 8873, 8874, 8876, 8877,
8881, 8882,
126
CA 02528853 2005-12-23
8883, 8885, 8887, 8888, 8889, 8890, 8892, 8894, 8896, 8897, 8899, 8902, 8785,
8903, 8904,
8905, 8906, 8907, 8908, 8911,
Detection: 8740, 8743, 8 746, 8765, 8774, 8781, 8792, 8795, 8811, 8817, 8822,
8825,
8834, 8839, 8849, 8858, 8860, 8862, 8875, 8886, 8910,
SEQ ID NO: 21
left Primer: 8913, 8917, 8920, 8923, 8924, 8929, 8931, 8934, 8935, 8936,
right Primer: 8914, 8916, 8918, 8921, 8925, 8927, 8928, 8930, 8932, 8933,
8937,
betection: 8915, 8919, 8922, 8926,
SEQ ID NO: 32
left Primer: 8938, 8944, 8945, 8948, 8952, 8953, 8954, 8957, 8958, 8961, 8962,
8965,
8967, 8969, 8970, 8971, 8972, 8977,
right Primer 8939, 8941, 8942, 8943, 8946, 8949, 8950, 8951, 8955, 8960, 8963,
8964,
8966, 8968, 8974, 8975, 8976, 8978, 8979,
Detection: 8940, 8947, 8956, 8959, 8973,
SEQ ID NO: 33
left Prnner: 8980, 8984, 8988, 8989, 8998, 9001, 9002, 9005, 9007, 9008, 9012,
9013,
9016, 9019, 9020, 9022, 9023, 9025, 9028, 9029, 9032, 9033, 9034, 9035y 9037,
9038, 9044,
9046, 9047, 9048, 9049, 9054,
right Primer 8981, 8983, 8985, 8987, 8990, 8992, 8993, 8994, 8995, 8997, 8999,
9003,
9004, 9006, 9009, 9010, 9011, 9014, 9017, 9018, 9021, 9024, 9026, 9030, 9036,
9039, 9041,
9042, 9043, 9045, 9050, 9051, 9052, 9053,
Detection: 8982, 8986, 8991, 8996, 9000, 9015, 9027, 9031, 9040,
SEQID NO:34
left Primer. 9055, 9058, 9060, 9063, 9066, 9067, 9068, 9069, 9070, 9071, 9074,
9075,
9077, 9078, 9079, 9080, 9082, 9084, 9088, 9089, 9090, 9099, 9103, 9104, 9105,
9107, 9111,
127
CA 02528853 2005-12-23
right Primer: 9056, 9061, 9064, 9072, 9081, 9083, 9085, 9086, 9087, 9091,
9092, 9093,
9094, 9095, 9096, 9097, 9098, 9100, 9102, 9106, 9108, 9109, 9110, 9112,
Detection: 9057, 9059, 9062, 9065, 9073, 9076, 9101,
SEQID NO:24
left Primer: 9113, 9116, 9118, 9125, 9126, 9128, 9129, 9130, 9136, 9141, 9143,
9148,
9150, 9153, 9157, 9158, 9159, 9163, 9164, 9165, 9167, 9168, 9171, 9173, 9177,
9180, 9181,
9182, 9183, 9185, 9187, 9188, 9189, 9191, 9192, 9193, 9194, 9197, 9198, 9200,
9206, 9207,
9208, 9209, 9211, 9216, 9218, 9219, 9220, 9221, 9222, 9223, 9226, 9228, 9229,
9230, 9231,
9232, 9233, 9234, 9237, 9238, 9240, 9243, 9246, 9248, 9253, 9256, 9257, 9260,
9263, 9264,
9266, 9269, 9271, 9279, 9280, 9281, 9282, 9283, 9284, 9290,
right Pri.mer: 9114, 9119, 9121, 9122, 9123, 9124, 9127, 9131, 9133, 9134,
9135, 9137,
9138, 9139, 9140, 9142, 9144, 9146, 9147, 9149, 9151, 9152, 9154, 9155, 9156,
9160, 9162,
9166, 9169, 9172, 9174, 9175, 9176, 9178, 9184, 9186, 9190, 9195, 9199, 9201,
9202, 9203,
9204, 9205, 9212, 9214, 9215, 9217, 9224, 9227; 9235, 9239, 9241, 9244, 9247,
9249, 9251,
9252, 9254, 9258, 9261, 9265, 9267, 9270, 9273, 9274, 9275, 9276, 9277, 9278,
9285, 9286,
9287, 9288; 9289, 9291,
Detection: 9115, 9117, 9 120, 9132, 9145, 9161, 9170, 9179, 9196, 9210, 9213,
9225,
9236, 9242, 9245, 9250, 9255, 9259, 9262, 9268, 9272,
SEQ ID NO: 35
left Primer: 9292, 9295, 9297, 9300, 9302, 9303, 9304, 9307, 9308, 9310, 9311,
9313,
9319, 9321, 9323, 9324, 9328, 9333, 9334, 9337, 9338, 9339, 9342, 9344, 9348,
9351, 9352,
9354, 9355, 9357, 9360, 9367, 9371, 9374, 9377, 9379, 9383, 9384, 9385, 9388,
9389, 9390,
9393, 9394, 9398, 9401, 9402, 9406, 9408, 9409, 9410, 9413, 9415, 9416, 9417,
9418, 9419,
9421, 9422, 9425, 9429, 9433, 9434, 9435, 9436, 9437, 9439, 9440, 9446, 9452,
9453, 9454,
9455, 9457, 9458, 9459, 9462, 9466, 9467, 9468, 9470, 9474, 9479, 9482, 9487,
9488, 9489,
9492, 9495, 9497, 9499, 9500, 9504, 9505, 9508, 9513, 9514, 9519, 9522, 9523,
9524, 9526,
9528, 9532, 9533, 9534, 9535, 9537, 9538, 9541, 9542, 9543, 9545, 9546, 9548,
128
CA 02528853 2005-12-23
right Primer: 9293, 9296, 9298, 9301, 9305, 9309, 9312, 9314, 9316, 9317,
9318, 9320,
9322, 9325, 9326, 9327, 9329, 9331, 9332, 9335, 9336, 9340, 9345, 9347, 9349,
9353, 9356,
9358, 9361, 9363, 9364, 9365. 9368, 9370, 9372, 9375, 9376, 9378, 9380, 9382,
9386, 9391,
9395, 9397, 9399, 9400, 9403, 9405, 9407, 9411, 9412, 9414, 9420, 9423, 9424,
9426, 9428,
9430, 9431, 9432, 9438, 9441, 9443, 9444, 9445, 9447, 9448, 9449, 9450, 9451,
9456, 9460,
9461, 9464, 9465, 9469, 9471, 9473, 9475, 9477, 9478, 9480, 9483, 9485, 9490,
9493, 9494,
9496, 9498, 9501, 9502, 9503, 9506, 9509, 9511, 9512, 9515, 9516, 9517, 9518,
9520, 9521,
9525, 9527, 9529, 9530, 9536, 9539, 9544, 9547, 9549, 9550,
Detection: 9294, 9299, 9 306, 9315, 9330, 9341, 9343, 9346, 9350, 9359, 9362,
9366,
9369, 9373, 9381, 9387, 9392, 9396, 9404, 9427; 9442, 9463, 9472, 9476, 9481,
9484, 9486,
9491, 9507, 9510, 9531, 9540,
SEQ ID NO: 25
left Primer: 9551, 9555, 9556, 9557, 9559, 9560, 9563, 9564, 9565, 9566, 9569,
9570,
9573, 9577, 9578, 9579, 9580, 9585, 9586, 9587, 9588, 9591, 9593, 9596,
right Primer. 9552, 9554, 9558, 9561, 9567, 9571, 9574, 9576, 9581, 9583,
9584, 9589,
9592, 9594, 9595,
Detection: 9553, 9562, 9568, 9572, 9575, 9582, 9590, 9597,
SEQ ID NO: 36
left Primer: 9598, 9602, 9604, 9605, 9608, 9612, 9613, 9614, 9617, 9618, 9621,
9622,
9625, 9626, 9627, 9632, 9633, 9638, 9640, 9643, 9644, 9645, 9646, 9651, 9654,
9656, 9657,
9658, 9661, 9662, 9665, 9666, 9667, 9668, 9672, 9673, 9675, 9676, 9677, 9680,
9683, 9686,
9688, 9691, 9694, 9698, 9699, 9702, 9705, 9707, 9710, 9711, 9715, 9719, 9720,
9722, 9723,
9726, 9729, 9732, 9733, 9735, 9737, 9738, 9741, 9744, 9745, 9746, 9747, 9748,
9749, 9750,
9754, 9755, 9756, 9758, 9759, 9768, 9771, 9772, 9776, 9777, 9781, 9782, 9784,
9787, 9789,
9790, 9792, 9796, 9797, 9800, 9805, 9806, 9812, 9815, 9818, 9821, 9823, 9825,
9827, 9831,
9832, 9835, 9837, 9839, 9841, 9842, 9844, 9845, 9851, 9853, 9854, 9855, 9857,
9860, 9862,
9863, 9865, 9871, 9873, 9875, 9878, 9879,
129
CA 02528853 2005-12-23
right Primer: 9599, 9601, 9603, 9606, 9607, 9609, 9611, 9615, 9619, 9623,
9624, 9628,
9630, 9631, 9634, 9635, 9637, 9639, 9641, 9647, 9649, 9650, 9652, 9653, 9659,
9660, 9663,
9664, 9670, 9671, 9679, 9682, 9684, 9687, 9690, 9692, 9693, 9695, 9697, 9700,
9701, 9703,
9706, 9708, 9712, 9713, 9714, 9716, 9717, 9718, 9721, 9724, 9727, 9730, 9734,
9739, 9742,
9751, 9753, 9757, 9760, 9761, 9762, 9763, 9764, 9765, 9766, 9767, 9769, 9773,
9775, 9778,
9780, 9783, 9786, 9788, 9791, 9793, 9794, 9795, 9798, 9801, 9803, 9804, 9807,
9808, 9809,
9810, 9811, 9813, 9816, 9817, 9819, 9820, 9822, 9826, 9828, 9830, 9833, 9836,
9838, 9840,
9843, 9846, 9847, 9848, 9849, 9850, 9852, 9856, 9858, 9859, 9861, 9864, 9866,
9867, 9868,
9869, 9870, 9872, 9876, 9877, 9880, 9881,
Detection: 9600, 9610, 9 616, 9620, 9629, 9636, 9642, 9648, 9655, 9669, 9674,
9678,
9681, 9685, 9689, 9696, 9704, 9709, 9725, 9728, 9731, 9736, 9740, 9743, 9752,
9770, 9774,
9779, 9785, 9799, 9802, 9814, 9824, 9829, 9834, 9874,
SEQ ID NO: 28
left Primer. 9882, 9886, 9890, 9891, 9894, 9900, 9902, 9904, 9908, 9911, 9912,
9915,
9917, 9919, 9923, 9925, 9928, 9935, 9936, 9940, 9943, 9949, 9953, 9954, 9956,
9957, 9958,
9962, 9963, 9964, 9968, 9969, 9975, 9978, 9981, 9988, 9989, 9990, 9991, 9992,
9995, 9997,
10011, 10012, 10014, 10017, 10018, 10031,10032,10033,10034, 10035,10037,
10041, 10042,
10046, 10050, 10054, 10058, 10059,
right Primer: 9883, 9885, 9887, 9889, 9892, 9893, 9895, 9896, 9897, 9898,
9899, 9901,
9903, 9905, 9907, 9909, 9913, 9914, 9916, 9918, 9920, 9921, 9922, 9924, 9926,
9929, 9931,
9932, 9933, 9934, 9937, 9938, 9939, 9941, 9942, 9944, 9945, 9946, 9947, 9948,
9950, 9952,
9955, 9959, 9960, 9961, 9966, 9967, 9970, 9972, 9973, 9974, 9976, 9979, 9980,
9982, 9983,
9984, 9985, 9986, 9987, 9993, 9994, 9996, 9998, 9999, 10000, 10001, 10002,
10003, 10004,
10005, 10006, 10007, 10008, 10009, 10010, 10013, 10015, 10016, 10019, 10020,
10021, 10022,
10023, 10025, 10026, 10027, 10028, 10030, 10036, 1003 8, 10040, 10043, 10044,
10045, 10047,
10048, 10049, 10051, 10053, 10055, 10056, 10057, 10060,
Detection: 9884, 9888, 9906, 9910, 9927, 9930, 9951, 9965, 9971, 9977, 10024,
10029,
10039, 10052,
130
CA 02528853 2005-12-23
SEQ ID NO: 37
left Primee 10061, 10064, 10067, 10068, 10070, 10071, 10072, 10074, 10079,
10081,
10083,10088,10091,10095, 10096,10097,10101,10103,10105,10106, 10109, 10110,
10111,
10113,10116,10118,10120,10122,10125,10126,10127, 10131,
10134,10137,10138,10144,
10147,10149,10151,10152,10155,10156, 10159,10160,10169,10172, 10174,10175,
10178,
10181, 10186,10188, 10189, 10190, 10191, 10192,10196,10199, 10203,
10204,10210,10211,
10216,10218,10221, 10224,10225, 10226,10229, 10230, 10231, 10232, 10234,
10239, 10241,
10244,10252,10253, 10257,10258, 10259,10261,10266, 10272, 10276,
right Primer. 10062, 10065, 10069, 10075, 10077, 10078, 10080, 10082, 10084,
10086,
10087, 10089, 10090, 10092, 10093, 10094, 10098, 10099, 10100, 10102, 10104,
10107, 10112,
10114,10117,10119, 10121, 10123, 10124, 10129, 10130,10132,10133, 10135,10139,
10140,
10 141, 10142, 10143, 10145,10148, 10150, 10153, 10154, 10157, 10161, 10163,
10164, 10165,
10166, 10167, 10170, 10173, 10176, 10177, 10179, 10182, 10183, 10184, 10187,
10193, 10194,
10195, 10197, 10198, 10200, 10201, 10202, 10205, 10206, 10207, 10209, 10212,
10213, 10214,
10215, 10219, 10220, 10222, 10223, 10227, 10228, 10233, 10235, 10236, 10237,
10240, 10242,
10243, 10245, 10247, 10248, 10249, 10251, 10254, 10255, 10256, 10260, 10262,
10263, 10264,
10265, 10267, 10268, 10269, 10270, 10271, 10273, 10274, 10275, 10277, 10278,
Detection: 10063, 10066, 10073, 10076, 10085, 10108, 10115, 10128, 10136,
10146,
10 158, 10162, 10168, 10171, 10180, 10185, 10208, 10217, 10238, 10246, 10250,
SEQ ED NO: 26
left Primer: 10279, 10282, 10285, 10287, 10291, 10292, 10297, 10299, 10301,
10304,
10306, 10308, 10310, 10314, 10317, 10318, 10320, 10322, 10327, 10329, 10330,
10333, 10335,
10336, 10337, 10340, 10342, 10348, 10350, 10351, 10354, 10358, 10359, 10360,
10362, 10366,
10369, 10372, 10373, 10374, 10376, 10378, 10383, 10385, 10391, 10398, 10403,
10407, 10410,
10413, 10415, 10419, 10422, 10425, 10427,10428,10429,10430, 10432, 10433,
10434, 10437,
1043 8, 10439, 10442, 10443, 10445, 10446, 10447, 10448, 10452, 10453, 10454,
10455, 10456,
10460, 10463, 10466, 10467, 10469, 10470, 10473, 10475, 10477, 10480, 10484,
10488, 10490,
131
CA 02528853 2005-12-23
10494, 10495, 10497, 10501, 10502, 10503, 10507, 10508, 10510, 10517,
10518,10520, 10522,
10524, 10525, 10528, 10529, 10533, 10536, 10537, 10540, 10541, 10545, 10547,
10551, 10553,
10556,10559,10560, 10563,10564,10565,10568,10571,10574, 10576,10579,10581,
10583,
10588, 10589, 10590, 10592, 10593, 10596, 10597, 10601, 10602, 10603, 10605,
10607, 10609,
10610, 10612, 10613, 10617, 10618, 10621, 10624,10627, 10630, 10632, 10634,
10637, 10638,
10639, 10640, 10641, 10643, 10645, 10647, 10653, 10655, 10657, 10659, 10661,
10662, 10663,
10664,10665,10666,10667,10668, 10669,10672,10673,10674,10675,
right Primer 10280, 10283, 10286, 10288, 10290, 10293, 10295, 10298, 10300,
10302,
10305, 10307, 10309, 10311, 10312, 10313, 10315, 10319, 10321, 10323,
10325,10326,10328,
10331, 10338, 10341, 10343, 10344, 10345, 10346, 10347, 10349, 10352, 10355,
10356, 10357,
10361, 10363, 10365, 10367, 10370, 10375, 10377, 10379, 10380, 10381, 10382,
10384, 10386,
10387, 10388, 10390, 10392, 10394, 10395, 10399, 10401, 10402, 10404, 10406,
10408, 10411,
10412, 10414, 10416, 10417, 10418, 10420, 10423, 10426, 10431, 10435, 10440,
10444, 10449,
10451, 10457, 10459, 10461, 10462, 10464, 10465,10468, 10471, 10476, 10478,
10481, 10483,
10485, 10487, 10489, 10491, 10492, 10493, 10498, 10499, 10500, 10504, 10506,
10509, 10511,
10513, 10514, 10515, 10516, 10519, 10521, 10523,10526,10530, 10531, 10532,
10534, 10535,
10538,10539, 10542, 10543,10544, 10546,10548,10550,10552, 10554, 10557,
10558,10561,
10566, 10569, 10572, 10573, 10577,10578,10580,10582,10584, 10586, 10594,10598,
10600,
10604,10606, 10611, 10614,10616, 10619, 10622,10625,10626, 10628, 10631,10633,
10635,
10642, 10644, 10646,10648,10649, 10650, 10651, 10652, 10654, 10656, 10658,
10660, 10670,
10671, 10676,
Detection: 10281, 10284, 10289, 10294, 10296, 10303, 10316, 10324, 10332,
10334,
10339, 10353, 10364, 10368, 10371, 10389, 10393, 10396, 10397, 10400, 10405,
10409,10421,
10424, 10436, 10441, 10450, 10458, 10472, 10474, 10479, 10482, 10486,
10496,10505, 10512,
10527, 10549, 10555, 10562, 10567, 10570, 10575, 10585,10587, 10591,
10595,10599, 10608,
10615,10620,10623,10629, 10636,10677,
SEQ ID xo: 38
left Primer: 10678,10684,
132
~~1
'fi0601 'Z0601'66801 '1.6801 'M01 '06801 '88801 '98801 '~8801 '18801
'08801'SL801 '~L801
'11.801 '01.801 '89801 '59801 'V9801 '~9801 'Z9801 '19801 '09801 '6S80i 'L5801
'tS801 '15801
'09801 'M0I 'Sfi80i 'ZV801 '1"01 'Oti801 '65801 '8~801 'L~80T 'S~80T'~~801
'I~801 't~Z80i
'ZZ801 '0Z801 '91801 'ST801 'V1801 '~1801'ZT801 '11801 '60801 'L080T '90801
'S0801 'b0801
'Z0801 '00801'66LOT '86L01 '176L0T '~6L0T'Z6L01'06L0T '68L0I '88L0I 'b8L0i
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'8LL0T '91,L01 'ELLOI '0LL0T 'L9L0T 'S9LOI '~9L0T 'Z9L0i '19L01 'LSLOI 'SSLOI
'ZSLOI 'OSLOT
'LbL01 'Ti,LOi '6~L0I '8~L0I 'L~L0T 'S~L0I '~~L0I 'Z~LOT '0~L01 'LZLOI '~ZL01
'6TL0I '81L0T
'LILOT '91L0I 't+IL01 '0TL0I 'LOLOT 'SOLOI 'ZOL01 '00L0I 'L6901 116901 =U!M UP
'~6011'580I1 '18011 '61.OTT '8L0TT 'ZL01I'ILOIT '0L0TT 'L901I '99011 't,9011
'Z90T[
'19011 10901I 'SSOTT 'ZSOT1 '8ti0TT '9ti0TI 'S"Ti '~bOiT 'ZbOTI 'IvOiT '060II
'9~0iT '~~OTT OZ
'SZOT I 'OZO T I '91011 '~I01T '600 i 1 '9001 T '100I 1 '000I 1 '66601 'L6601
'S6601 'Z6601 '88601
'S8601 't,8601 '18601 '81.601 'LL601 'SL601 'ZL601 '89601 'L9601 'Z9601 '6S601
'8S601 695601
"S5601 '~5601 '0601 '9M1 'tfi60T '~b601 'Zb601 'IV601 '8E601 'ti~601 '~~601
'Z~601 '0~60I
'9Z601 '61601 '91601 'tr160i '11601 '01601 '60601 '1.0601 '50601 '~0601
'1.0601 '00601 '96801
'~6801 'Z6801 '68801 'S8801 118801 'Z8801'6L801 '81.801 'LL80I 'Y1.801 'ZL801
'69801 '9980i ST
'8S801 '95801 '~S801 '61801 '8b801 'LV80T '1,b801 'Eti801 'V5801 '0~801 '6Z801
'LZ801 '9Z801
'~Z80T '1Z801 461801 '81801 '1.1801 '80801 '10801 '961.01 '161.01 '981.01
'~8L01 '181.01 '08L01
'SLLOT 'ZLLOI '69L0T '89L01 'i-9L0T '09L0T '6SLOi'8SLOt 'KL01 'ISLOI '6fiL0i
'81,L0t '9bL0I
'MOT '1tL0T '5tL01 'ZbLOi '0f'L0T 'KL01 '6ZL0T '8ZL0T '9ZL0I 'SZLOT 'bZLOi
'ZZLOI 'TZLOT
'OZLOT '~ILOT 'ZIL01 'ITL01 '60L0I 'VOL0I '~OLOI '66901 '96901 '~6901 :num.d
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LZ:OAid.Ia88
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'68901 '88901 '48901 '58901 '~8901 'Z8901 '18901 '61.901 =Mud 311B?1
~Z-ZT-SOOZ ~588ZSZ0 Fi'J
CA 02528853 2005-12-23
10906, 10908, 10912, 10915, 10917, 10920, 10922, 10924, 10925, 10927, 10928,
10929, 1093.1,
10935, 10937, 10939, 10945, 10948, 10950, 10951, 10952, 10954, 10957, 10960,
10963, 10965,
10966,10969,
10970,10971,10973,10976,10979,10980,10982,10986,10987,10989,10991,
10993, 10996, 10998, 11002, 11004, 11005, 11007, 11010, 11012, 11014, 11015,
11017, 11019,
11021, 11022, 11023, 11026, 11027, 11028, 11029, 11030, 11031, 11032, 11034,
11037, 11039,
11044, 11047, 11049, 11050, 11051, 11053, 11054, 11056, 11058, 11059, 11063,
11066, 11068,
11069, 11073, 11074, 11075, 11076, 11077, 11080, 11082, 11083, 11084, 11086,
11087, 11088,
11089,11090, 11091, 11092,11094,
Detection: 10695, 10698, 10701, 10706, 10708, 10715, 10731, 10736, 10753,
10756,
10766, 10771, 10774, 10777, 10785, 10787, 10795, 10797, 10803, 10810, 10825,
10828, 10832,
10836, 10852, 10855, 10867, 10876, 10887, 10891, 10895, 10898, 10913, 109,18,
10921, 10923,
10936, 10940, 10949, 10961, 10964, 10974, 10983, 10990, 10994, 11003, 11008,
11011, 11018,
11024, 11035, 11038, 11057, 11095,
SEQ ID NO: 40
left Primer: 11376, 11381, 11382, 11383, 11384, 11387, 11397, 11401, 11405,
11408,
11414,11418, 11421, 11422, 11424,11427,11429, 11430,
right Primer: 11377, 11379, 11380, 11385, 11386, 11388, 11390, 11391, 11392,
11394,
11395,11396, 11398,11399,11400,11402, 11403, 11404, 11406,11409, 11410, 11411,
11412,
11415, 11416, 11417, 11419,11423,11425,11428, 11431, 11432,11433,
Detection: 11378, 11389, 11393, 11407,11413, 11420, 11426,
Light cycler dnal probe assays:
SEQ ID NO: 41
left Primer 11434, 11438, 11442, 11443, 11447, 11449, 11450, 11452, 11456,
11459,
11463, 11464, 11467, 11468, 11472, 11477,11480, 11481, 11482, 11483, 11484,
11488, 11489,
11493,11498, 11502, 11504, 11505,11506, 11507, 11508,11509, 11512, 11513,
11514, 11517,
11520, 11522, 11523, 11524, 11526, 11528, 11532, 11534,
right Primer: 11435, 11439, 11444, 11445, 11446, 11448, 11451, 11453, 11457,
11458,
134
CA 02528853 2005-12-23
11460,11461,11462,11465,11466,11469,11473,11474,11475,11476,11478,11479,11485,
11486,11487,11490,11494,11497,11499,11500,11501, 11503,11510, 11511, 11515,
11518,
11519,11521, 11525, 11527,11529,
Detection:11436,11440,11454, 11470, 11491,11495,11530,
Anchor: 11437,11441,11455,11471,11492,11496,11516,11531,11533,
SEQ ID NO: 5
left Primer. 11535, 11539, 11540, 11541, 11548, 11550, 11551, 11552, 11555,
115S6,
11557,11560, 11561,11562,11568,11569,11571, 11572, 11573,11577,
right Prinner: 11536, 11542, 11545, 11546, 11547, 11549, 11553, 11554, 11563,
11566,
11570, 11574,
Detection:11537,11543,11558,11564,11575,
Anchor: 11538,11544,11559, 11565, 11567, 11576,
SEQ ID NO: 6
left Primer. 11578,11585, 11586,11590, 11591, 11592,11595, 11596, 11597,
11600,
right Primer. 11579,11582,11587,11593, 11594,11598, 11601,
Detection: 11580,11583, 11588,
Anchor: 11581, 11584,11589, 11599,
SE.Q ID NO: 8
lefft Primer. 11602, 11606, 11607, 11608, 11609, 11616, 11618, 11622, 11626,
11628,,
11632, 11633,
right Primer 11603, 11610, 11613, 11614, 11615, 11617, 11619, 11623, 11627,
11629,
11634,
Detection:11604,11611,11620,11624,11630,
Anchor: 11605,11612,11621,11625,11631,
SEQ ID NO: 16
135
CA 02528853 2005-12-23
left Primer: 11635, 11640, 11641, 11642, 11643, 11647, 11648, 11654, 11655,
11656,
11657,11658,
right Primer:11636,11639,11644,11645,11646, 11649,11652,11653,
Detectian: 11637,11650,
Anchor: 11638,11651,
132
SEQIDNO:42
left Primer. 11659, 11663, 11664, 11665, 11666, 11670, 11671, 11672, 11673,
11677,
11681,11687, 11690,11694,11696, 11697, 11701,11705,11709,
11713,11719,11723,11729,
11733,11739,11744,11745,11748,11749,11751,11755,11759, 11763,
11770,11772,11774,
11776,11778,11782,11785,
right Primer 11660, 11667, 11674, 11675, 11676, 11678, 11682, 11685, 11686,
11688,
11689,11691, 11692,11693, 11695, 11698, 11699, 11700,11702, 11706, 11710,
11716,11720,
11724,11725,11727,11728,11730, 11731, 11732,11734,11735, 11736,
11737,11738,11740,
-11743,11746, 11747, 11750,11752, 11756, 11761,11762, 11764,11765, 11767,
11768, 11769,
11773,11775, 11777,11779,11783, 11784,
Detection: 11661, 11668, 11679, 11683, 11703, 11707, 11711, 11714, 11717,
11721,
11726,11741, 11753,11757,11766,11771, 11780,
Anchor: 11662, 11669, 11680, 11684, 11704, 11708, 11712, 11715, 11718, 11722,
11742,11754,11758,11760, 11781,
SEQIDNO:14
left Primer: 11786,11790,11791,11792,
right Primer: 11787, 11793,
Detecfion: 11788,11794,
Anchor: 11789,11795,
SEQ ID NO: 43
left Primer. 11796, 11800, 11802, 11803, 11805, 11810, 11818, 11819, 11823,
11824,
136
CA 02528853 2005-12-23
11827, 11828, 11829,11833,11836,11837, 11839, 11840, 11847, 11850, 11852,
11857, 11860,
11864,11866,
right Primer: 11797, 11801, 11804, 11806, 11809, 11811, 11814, 11815, 11817,
11820,
11825, 11830, 11835, 11841, 11844, 11845, 11846, 11848, 11851, 11853, 11856,
11858, 11861,
11862,11863,11865,
Detection: 11798, 11807, 11812,11816,11821, 11831,11842, 11854,
Anchor 11799, 11808, 11813, 11822, 11826, 11832, 11834, 11838, 11843, 11849,
11855, 11859,
SEQ ID NO: 15
left Prixner: 11867,11871,11876,11878,11882,11888,11891,11894,
right Primer: 11868, 11872, 11875, 11877, 11879, 11883, 11886, 11887, 11889,
11890,
11892,11893,11895,
Detection: 11869,11873, 11880,11884, 11896,
Anchor: 11870, 11874, 11881, 11885, 11897,
SEQ ID NO:4
left Primer. 11898, 11902, 11903, 11904, 11908, 11909, 11911, 11912, 11913,
11914,
11915, 11917, 11918,11922, 11926,11927, 11930,11932, 11933, 11937,
11941,11942, 11946,
11947,11948,11949,11950,11951,11952,11955,11959,11968, 11972, 11976,11978,
11979,
11980, 11981, 11986, 11988, 11989,11991, 11992, 11995, 11997, 11998, 12001,
12002, 12004,
12005,
right Primer: 11899, 11905, 11910, 11916, 11919, 11923, 11924, 11925, 11928,
11929,
11931,11934,11938,11943,11953, 11954, 11956, 11960, 11963, 11964, 11967,
11969, 11973,
11977,11982, 11985,11987, 11990, 11993,11994, 11996,11999,12000, 12003,
Detection: 11900, 11906, 11920, 11935, 11939, 11944, 11957, 11961, 11965,
11970,
11974,11983,
Anchor. 11901, 11907, 11921, 11936, 11940, 11945, 11958, 11962, 11966, 11971,
11975, 11984,
137
CA 02528853 2005-12-23
SEQ ID NO: 7
left Primer: 12006, 12011, 12015, 12016, 12017, 12021, 12022, 12023, 12024,
12025,
12026,12027,12031,12034, i2042,12043, 12045,12049,12050, 12051, 12054,12062,
12063,
12064,12066,12067, 12068, 12070,12072, 12076,12082,12085, 12086, 12089, 12090,
12093,
12094,12095,12096, 12097,12098,12101, 12103,12107,12108,12111,12112,12114,
12118,
12119, 12120,12128,
right Primw. 12007, 12010, 12012, 12020, 12028, 12032, 12033, 12035, 12038,
12039,
12044, 12046, 12052, 12055,12056,12057, 12058,12059,12060,12061, 12065, 12069,
12071,
12073,12077,12080,12081,12084,12087,12088,12091,12099,12100,12102,12104,12109,
12110,12113, 12115,12121, 12122,12123, 12124, 12125,12127, 12129,
Detection: 12008, 12013, 12018, 12029, 12036, 12040, 12047, 12074, 12078,
12083,
12105,12116,
Anchor. 12009, 12014, 12019, 12030, 12037, 12041, 12048, 12053, 12075, 12079,
12036,12092,12106,12117,12126,
SEQ ID NO:1
left Primer: 12130, 12135, 12136, 12141, 12144, 12147, 12148, 12149, 12153,
12159,
12160,
right Primer: 12131, 12134, 12137, 12138, 12139, 12140, 12142, 12143, 12145,
12146,
12150,12154,12157,12158,12161,12162,12163,
Detection: 12132,12151,12155,
Anchor: 12133, 12152, 12156,
SEQ II1 NO: 45
left Primer. 12164, 12168, 12171, 12176, 12181, 12187, 12190, 12194, 12196,
12197,
12199,12201,12202, 12209,12213,12216, 12220,12221,12223,12225, 12226,12231,
12233,
12239,12243,12244, 12250, 12254, 12256, 12257,12259, 12266, 12270, 12271,
12272, 12274,
12276,12277,12278, 12280,12284, 12285, 12288,12289,12290, 12291, 12295,12296,
12302,
138
CA 02528853 2005-12-23
12308, 12313, 12317, 12321, 12322, 12323, 12324, 12325, 12327, 12328, 12330,
12331, 12332,
12336,12337, 12339,12343,12346,12349,12353,12354,12355,12359,12361,12363,
12367,
12369, 12370, 12372, 12376, 12377, 12379, 12380, 12381, 12382, 12383, 12385,
12389, 12393,
12398, 12400, 12402, 12403, 12406, 12408, 12409, 12410, 12412, 12415, 12417,
12419, 12422,
12424,12429, 12430,12432,12433,12435, 12436,12437, 12441,12445,12448, 12449,
12454,
12458,12459, 12460,
right Primer. 12165, 12169, 12170, 12172, 12175, 12177, 12180, 12182, 12185,
12186,
12188, 12191, 12195, 12198, 12200, 12203, 12206, 12207, 12208, 12210, 12214,
12217, 12222,
12224,12227, 12228, 12229, 12230, 12232, 12234, 12237,12238, 12240, 12241,
12242, 12245,
12248,12249,12251,12255,12258,12260,12262,12263, 12264,12265,12267, 12268,
12269,
12273, 12279, 12281, 12286, 12287, 12292, 12297, 12300, 12303, 12306, 12307,
12309, 12312,
12314,12318,12326,12333,
12338,12340,12341,12342,12345,12347,12348,12352,12356,
12360,12362,12364,12371,12373,12378,
12386,12390,12394,12397,12401,12404,12405,
12411, 12413, 12418, 12420, 12423, 12425, 12428, 12438,12439,12440, 12442,
12443, 12446,
12450,12453,12455,
Detection: 12166, 12173, 12178, 12183, 12189, 12192, 12204, 12211, 12218,
12235,
12246, 12252, 12282, 12293,12298,12301, 12304,12310,12315,12319, 12334, 12350;
12357,
12365, 123.74, 12387, 12391,12395, 12416, 12426, 12444, 12451,12456,
.Anchor. 12167, 12174, 12179, 12184, 12193, 12205, 12212, 12215, 12219, 12236,
12247,12253,12261,12275,12283,12294, 12299,12305, 12311,12316,12320,12329,
12335,
12344,12351, 12358, 12366,12368, 12375,12384, 12388,12392,12396, 12399,12407,
12414,
12421, 12427, 12431, 12434, 12447, 12452, 12457,
SEQIDNO:9
left Primer: 12461, 12465, 12467, 12468, 12472, 12474, 12477, 12480, 12484,
12486,
12488,12489,12490,12493,12495,12499,12501,12504,12506,
right Primer. 12462, 12469, 12475, 12478, 12481, 12483, 12485, 12487, 12492,
12494,
12496, 12497, 12498,12500, 12502,12505,
Detection: 12463,12470,12473,12482,
139
CA 02528853 2005-12-23
Anchor. 12464, 12466,12471,12476,12479,12491,12503,
SEQ ID NO: 46
left Primer: 12507, 12511, 12517, 12520, 12522, 12523, 12524, 12528, 12530,
12534,
12535, 12536, 12537, 12541, 12542, 12547, 12550, 12551, 12552, 12554,
12555,12560,12564,
12565,12569, 12571,12574,12579,12581,12586,12590, 12586,12590,12594,12596,
12600,
12601,12602,12604,12610,12613,12614,12615,12616,12620,12626,12630,
right Ffimer: 12508, 12512, 12515, 12518, 12521, 12525, 12529, 12531, 12538,
12543,
12546, 12548, 12549, 12553, 12556, 12559, 12561, 12566, 12570, 12572, 12573,
12575, 12578,
12582, 12585, 12587, 12595, 12597, 12599, 12603, 12605, 12608, 12609, 12611,
12617, 12618,
12621,12624,12625,12627,
Detection: 12509, 12513, 12516, 12514, 12532, 12539, 12544, 12557, 12562,
12567,
12576,12583, 12588,12591,12606,12612,12622,12628,
Anchor: 12510, 12514, 12519, 12526, 12527, 12533, 12540, 12545, 12558, 12563,
12568,12577, 12580,12584,12589,12598,12607,12619,12623, 12629,
SEQ ID NO: 10
left Primer: 12631, 12636, 12637, 12638, 12639, 12643, 12647, 12648, 12652,
12654,
12660,12664,12666,12670,12672, 12675,12677, 12680,12686,12691, 12692, 12695,
12699,
12700, 12701, 12703, 12705, 12709, 12710, 12714, 12715, 12716, 12718, 12719,
12720, 12724,
12725, 12728, 12732, 12738, 12739, 12743, 12744, 12745, 12747, 12748, 12752,
12757, 12763,
12764, 12766, 12767, 12768, 12771, 12776, 12777, 12781, 12785, 12787, 12788,
12791, 12792,
12796, 12797, 12800, 12804, 12808, 12813, 12815, 12816, 12817, 12822, 12825,
12828, 12831,
12833,12834,12837, 12839, 12841, 12842,
right Primer: 12632, 12635, 12640, 12641, 12644, 12649, 12653, 12655, 12658,
12661,
12662, 12663, 12665, 12667, 12671, 12673, 12678, 12679, 12681, 12682, 12683,
12684, 12685,
12687, 12688, 12689, 12690, 12693,12694,12696,12702,12704,12706, 12712, 12713,
12717,
12721, 12726, 12729, 12730, 12731, 12733, 12736, 12737, 12740, 12746, 12749,
12750, 12751,
12753,12754, 12755, 12756, 12758,12760, 12761, 12762, 12765, 12769, 12770,
12772, 12=775,
140
CA 02528853 2005-12-23
12778,12782,12786, 12789,12790,12793,12794,12795, 12798, 12799,
12801,12805,12809,
12812,12814,12818,12821,12823,12824,12826,12827,12832,12835,12836,12838,12840,
12843,12844,
Detection: 12633, 12642, 12645, 12650, 12656, 12668, 12674, 12676, 12697,
12707,
12722,12734,12741,12759,12773,12779,12783,12802, 12806,12810,12819,12829,
Anchor: 12634, 12646, 12651, 12657, 12659, 12669, 12698, 12708, 12711, 12723,
12707, 12727, 12735, 12742, 12774, 12780, 12784,12803, 12807, 12811, 12820,
12830,
SEQ ID NO:11
left Prima: 12845, 12849, 12850, 12854, 12857, 12862, 12866, 12867, 12868,
12870,
12872, 12880,12881,12884, 12885,12889; 12890, 12892, 12896, 12898, 12899,
12903, 12907,
12910, 12913, 12919, 12920, 12921, 12923, 12928, 12932, 1293 8, 12942, 12945,
12948, 12950,
12951,12953, 12959,
right Prinner: 12846, 12851, 12855, 12858, 12861, 12863, 12869, 12871, 12873,
12876,
12877, 12878, 12879, 12882, 12883, 12886, 12887, 12888, 12891, 12893, 12894,
12895, 12900,
12901, 12902, 12904, 12908, 12909, 12911, 12912, 12914, 12917,
12918,12924,12927,12929,.
12933, 12936, 12937, 12939, 12943, 12949, 12954, 12957, 12958,12960,
Detection: 12847, 12852, 12859, 12864, 12874, 12905, 12915, 12925, 12930,
12934,
129940,12944, 12946,12955,
Anchor. 12848, 12853, 12856, 12860, 12865, 12875, 12897, 12906, 12916, 12922,
12926,12931,12935,12941,12930,12947, 12952,12956,
SEQ ID NO: 12
left Primer. 12961, 12965, 12969, 12975, 12977,
right Primer: 12962, 12966, 12967, 12968, 12970, 12971, 12972, 12973, 12974,
12976,
12978,12981,
Detectioa: 12963,12979,
Anchor. 12964, 12980,
141
CA 02528853 2005-12-23
SEQ ID NO: 13
left Primer: 12982, 12986, 12989, 12991, 12992, 12993, 12995, 12999, 13001,
right Primer.12983,12987,12996,12997,12998,13000,13002,
Detection: 12984, 13003,
Anchor: 12985, 12988, 12990, 12994, 13004,
SBQ IDNO:3
lefft Primer: 13005, 13009, 13013, 13017, 13019, 13025, 13029, 13030, 13032,
13033,
13034, 13037,13040, 13042,13043, 13044,13048,13049,13051, 13052, 13053,
13057,13062,
13064, 13065, 13066, 13067, 13072, 13073, 13076, 13078, 13082, 13083, 13085,
13087, 13088,
13089,13092,13096, 13100, 13101, 13102, 13105, 13108, 13109, 13110, 13114,
13117, 13118,
13124, 13129, 13131, 13132, 13138, 13144, 13146, 13147, 13148, 13152, 13156,
13159, 13164,
13167, 13168, 13170,13171,13172,13173,13174, 13184, 13185,
right Primer: 13006, 13010, 13014, 13018, 13020, 13021, 13022, 13023, 13024,
13026,
13031, 13035, 13036, 13038, 13039, 13041, 13045, 13050, 13054, 1305 8, 13061,
13063, 13068,
13069, 13070, 13071, 13074, 13075, 13077, 13079, 13084, 13086, 13090,
13091,13093,13097;
13104,13107,13111,13115,13119,13121,13122,13123, 13125,13126,13127,13128,
13130,
13133,13137,13139,13142, 13143,13145, 13149,13153,13157, 13158, 13160,13163,
13165,
13166, 13169, 13175, 13176, 13177, 13178, 13179, 13180, 13181,13182,13183,
13186, 13189,
13190,
Detection: 13007, 13011, 13015, 13027, 13046, 13055, 13059, 13080, 13094,
13098,
13112, 13120, 13134, 13140, 13150, 13154, 13161, 13187,
Anchor. 13008, 13012, 13016, 13028, 13047, 13056, 13060, 13081, 13095, 13099,
13103,13106,13113, 13116, 13135,13136, 13141,13151, 13155,13162, 13188,
SEQ ID NO: 47
leftPrimer: 13191,13195,13196,13197, 13199,13200,13204,13206,
right Primec 13192, 13198,13201,
Detection: 13193,13202,
142
CA 02528853 2005-12-23
Anchor:13194,13203,13205,
SEQID NO: 18
left Primer: 13207, 13212, 13213, 13226, 13228, 13231, 13235, 13240, 13245,
13247,
13248, 13250, 13254, 13256, 13258, 13264, 13265, 13268, 13269, 13273, 13274,
13277, 13279,
13280,13281, 13286,
right Primer. 13208, 13211, 13214, 13217, 13220, 13222, 13224, 13225, 13227,
13229,
13232,13233,13234,13237,13239,13241,13242,13?A4,13246,13249,13251,13255,13257,
13259, 13262,13263, 13266,13267,13270,13275,13276,13282,13285,
Detection: 13209,13215, 13218, 13236,13252, 13260,13271,13283,
Anchor: 13210, 13216, 13219, 13221, 13223, 13230, 13238, 13243, 13253, 13261,
13272,13278, 13284,
SEQ ID NO: 32
left Primer: 13641, 13645, 13646, 13649, 13650, 13652, 13653, 13654, 13655,
13658,
13659,
right Primer: 13642, 13647',13648,13656,
Detection: 13643,
.Anchor:13644,13651,13657,
SEQ ID NO: 33
left Primer: 13660,
right Primer: 13661,
Detection: 13662,
Anchor.13663,
SEQ ID NO: 34
left Primer: 13664, 13669, 13670, 13671, 13672, 13673, 13675, 13676, 13677,
13681,
13683, 13685, 13687, 13689, 13691, 13693, 13696,
143
CA 02528853 2005-12-23
night Primer: 13665, 13668, 13674, 13678, 13682, 13684, 13686, 13688, 13690,
13692,
13694,13695,
Detecc tion:13666, 13679,
Anchor: 13667,13680,
SEQ ID NO: 24
left Primer. 13697, 13701, 13702, 13703, 13709, 13713, 13714, 13715, 13719,
13721,
13722,13726,13728,13732,13735,13740,
right Primer: 13698, 13704, 13705, 13706, 13707, 13708, 13710, 13716, 13717,
13718,
13720,13723, 13724,13725,13727,13729,13733, 13734,13736,13739,13741, 13744,
Deteation:13699, 13711, 13730, 13737, 13742,
Anchor: 13700,13712,13731,13738,13743,
SEQ ID NO: 35
left Primer: 13745, 13749, 13751, 13752, 13753, 13754, 13758, 13762, 13765,
13766,
13768,13772, 13773,13774,13777,13778,13779,
13783,13785,13790,13791,13797,13802,
13803, 13805, 13806, 13807, 13809, 13812, 13814, 13816, 13817, 13818, 13819,
13820, 13823,
13824, 13826,13828,
right Primer: 13746, 13755, 13759, 13763, 13764, 13767, 13769, 13775, 13776,
13780,
13784,13786,13788, 13789, 13792, 13795, 13796,13798, 13800, 13804,13808,
13813, 13815,
13821,13822, 13829,
Detection: 13747, 13756, 13760,13781,13793,13801,13810,13830,
Anchor. 13748, 13750, 13757, 13761, 13770, 13771, 13782, 13787, 13794, 13799,
13811,13825, 13827,13831,
SEQID NO:36
left Primer 13832, 13837, 13841, 13844, 13845, 13846, 13849, 13854, 13855,
13856,
13857, 13861, 13865, 13869, 13871,13875, 13879, 13880,
right Primer: 13833, 13836, 13838, 13843, 13847, 13850, 13853, 13858, 13862,
13866,
144
CA 02528853 2005-12-23
13870,13872, 13876,13881,13882,
Detection: 13834, 13839, 13851, 13859, 13863, 13867, 13873, 13877,
Anchor: 13835,13840, 13842,13848,13852,13860,13864,13868, 13874, 13878,
SEQ ID NO: 28
left Primer: 13883, 13889, 13894, 13902, 13903, 13910, 13911, 13914, 13915,
13916,
13928,13929,13930,13931, 13935, 13936,13937, 13944,13947, 13948,13950,13960,
13961,
13962,13964, 13965,13966, 13968,13970,13971,13972,13976, 13978,
right Primer: 13884, 13887, 13890, 13893, 13895, 13898, 13900, 13904, 13905,
13906,
13907,13908, 13909,13912, 13913, 13917, 13920, 13921, 13922, 13923, 13924,
13925, 13926,
13932,13938,13941,13942,13943, 13945, 13946,13949, 13953, 13954, 13956,13957,
13958,
13959, 13963,13967, 13969, 13974, 13975,13977,13980,
Detection: 13885, 13886, 13891, 13896, 13901, 13918, 13927, 13933, 13939,
13951,
Anchor: 13886, 13888, 13892, 13897, 13899, 13901, 13919, 13934, 13940, 13952,
13955,13973, 13979,
SEQ ID NO: 37
left1'rimer: 13981,13988,13989, 13990, 13992,13994,13995, 13999,
right Primer: 13982, 13985, 13986, 13987, 13991, 13993, 13996, 14000, 14001,
14002,
14003,
Detection: 13983, 13997,
Anchor: 13984, 13998,
SEQ ID NO: 26
left Primer. 14004, 14009, 14014, 14017, 14020, 14024, 14026, 14029, 14035,
14036,
14040,14043,14045,14051,14057,14058,14059, 14063,14065,14066,14067,14068,
14069,
14070, 14071, 14073,14074,14078,14079,14080,14081, 14082, 14083, 14084, 14085,
14087,
14094,14095, 14100,14108,14109, 14113,14114,14118, 14120, 14121, 14122,
14130,14133,
14138, 14141, 14142, 14143, 14147,
145
CA 02528853 2005-12-23
right Primer: 14005, 14008, 14010, 14013, 14015, 14018, 14021, 14025, 14027,
14028,
14030, 14033,14034, 14037,14038,14039,14041,14042,14044,14046,
14049,14050,14052,
14055,14056, 14060,14064,14072,14077, 14086,14088,14089, 14090, 14091,14092,
14093,
14096,14097, 14098, 14099, 14101,14102,14103,14104,14105, 14106,
14107,14110,14115,
14123,14126,14127,14132,14134,14135,14136,14137,14139,14140,14144,14145,14148,
Ikteation: 14006, 14011, 14019, 14022, 14031, 14047, 14053, 14061, 14075,
14111,
14116, 14124, 14149,
Anchor. 14007, 14012, 14016, 14023, 14032, 14048, 14054, 14062, 14076, 14112,
14117, 14119, 14125, 14128, 14129, 14131,14146,14150,
SEQ ID NO: 27
left Primer: 14151, 14155, 14157, 14161, 14166, 14171, 14175, 14183, 14187,
14190,
14192,14194,14195, 14196, 14200,14201,
right Primer: 14152, 14156, 14158, 14162, 14165, 14167, 14170, 14172, 14176,
14179,
14180, 14182,14184, 14185, 14186, 14188, 14189, 14191, 14193, 14197,
14198,14199, 14202,
14203,
Detection:14153, 14159,14163,14168,14173,14177,
Anohor: 14154, 14160, 14164,14169, 14174, 14178, 14181,
SEQ ID NO: 31
left Primer. 14204, 14210, 14211, 14216, 14217, 14223, 14226, 14228, 14229,
14230,
14233, 14235, 14239,14241,14243, 14244, 14250,14254,14255, 14256, 14257,14260,
14261,
14262, 14267,14268,14269,14273, 14274, 14277,14281,14284, 14286, 14289, 14292,
14293,
14297, 14299,14301,14304,14305,14306,14312,14316,14320, 14322,14324, 14328,
14329,
14331, 14337, 14342, 14344, 14346, 14347,14349,14350,14353, 14356, 14361,
14363, 14364,
14365, 14366,
right Primer. 14205, 14208, 14212, 14215, 14218, 14221, 14224, 14225, 14227,
14231,
14236, 14240, 14242, 14245, 14246, 14247, 14251, 14263, 14266,
14270,14278,14282, 14283,
14285, 14287,14290, 14291, 14294, 14298, 14300,14302, 14303, 1430'],
14310,14313, 14317,
146
CA 02528853 2005-12-23
14323,14325,14327,14330,14334,14338,14340,14341,14345,14348,14351, 14352,
14354,
14355,14357,14358,14359,14360,14367,
Detection: 14206, 14213, 14219, 14237, 14248, 14252, 14258, 14264, 14271,
14275,
14279,14295,14308,14311,14314,14318,14332,14335,14339, 14343,
Anchor. 14207, 14209, 14214, 14220, 14222, 14232, 14234, 14238, 14249, 14253,
14259, 14265,14272, 14276,14280, 14288,14296, 14309, 14315, 14319, 14321,
14326, 14333,
14336,14362,
SEQIDNO:40
leR Primer. 14368,14372,14373,14375,14386,
right Frimer.14369, 14374, 14376, 14379, 14380, 14381, 14382, 14383, 14384,
14385,
Detection: 14370,14377,
Anchor: 14371, 14378,
In an alternative most preferred embodiment of the method, the subsequent
ampli$cation
of d) is carried out in the presence of blocktng oligonueleotides, as
described above. Said
blocking oligonucleotldes comprising a sequence having a length of at least 9
nucleotides which
hybridizes to a treated nucleic acid sequence according to one of SEQ ID
NOS:304 to SEQ ID
NO:535 and sequences complementary thereto, wherein the base sequence of said
oligomers
comprises at least one CpG, TpG or CpA dinucleotide. Step e) of the method,
namely the
detection of the speaific amplfficates indicative of the methylation status of
one or more CpG
positions according to SEQ ID NOS:l to SEQ ID NO:64 is carried out by means of
real-time
detection methods as desen'bed above.
In a further preferred embodimeat of the method the sequence(s) of the
blocking
oligonucleotides are selected from the group consisting of SEQ ID NOS:2114,
2119, 2124,
2125, 2127, 2132, 2133, 2135, 2137, 2138, 2139, 2140, 2141, 2142, 2143, 2144,
2146, 2147,
2148, 2149, 2150, 2151, 2152, 2153, 2154, 2155, 2156, 2157, 2158, 2159, 2160,
2031, 2082,
2087, 2089, 2094, 2095, 2096, 2097, 2099, 2107, 1980, 2018, 2022, 2023, 2026,
1939, 1966,
1973, 1974, 1975, 1926, 1930, 1931, 1932, 1933, 1934, 1903, 1917, 1921, 1834,
1896, 1805,
147
CA 02528853 2005-12-23
1819, 1821, 1823, 1824, 1825, 1826, 1827, 1828, 1829, 1770, 1782, 1785, 1786,
1788, 1789,
1790, 1792, 1795, 1796, 1797, 1798, 1799, 1800, 1748, 1759, 1760, 1761, 1762,
1763, 1764,
1765, 1724, 1741, 1597, 1609, 1610, 1611, 1612, 1613, 1614, 1615, 1616, 1617,
1618, 1619,
1620, 1621, 1622, 1623, 1624, 1625, 1626, 1627, 1628, 1629, 1630, 1631, 1633,
1634, 1635,
1636, 1637, 1638, 1639, 1640, 1641, 1642, 1643, 1644, 1645, 1646, 1647, 1648,
1649, 1650,
1651, 1652, 1653, 1654, 1655, 1656, 1657, 1658, 1659, 1660, 1661, 1662, 1663,
1668, 1684,
1685, 1686, 1687, 1688, 1689, 1690, 1691, 1692, 1693, 1694, 1695, 1696, 1697,
1698, 1699,
1700, 1701, 1706, 1712, 1713, 1715, 1716, 1717, 1718, 1719, 1571, 1579, 1581,
1584, 1585,
1586, 1587, 1588, 1591, 1592, 1537, 1555, 1556, 1557, 1558, 1559, 1560, 1561,
1562, 1563,
1564, 1565, 1566, 1452, 1455, 1456, 1457, 1458, 1463, 1514, 1515, 1516, 1517,
1518, 1519,
1524, 1525, 1526, 1527, 1528, 1529, 1531, 1532, 1372, 1423, 1424, 1425, 1427,
1432, 1433,
1434, 1435, 1436, 1437, 1440, 1441, 1442, 1443, 1444, 1445, 1446, 1447, 1343,
1358, 1359,
1360, 1361, 1362, 1363, 1364, 1365, 1366, 1367, 1310, 131 S, 1316, 1317, 1318,
1319, 1320,
1321, 1322, 1324, 1325, 1327, 1328, 1330, 1332, 1334, 1337, 1338, 1276, 1290,
1305, 1203,
1228, 1238, 1239, 1240, 1247, 1248, 1255, 1256, 1265, 1268, 1269, 1164, 1171,
1172, 1173,
1174, 1176, 1177, 1178, 1179, 1180, 1181, 1182, 1183, 1184, 1185, 1186, 1187,
1188, 1189,
1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, 2725, 2748, 2749, 2750,
2710, 2717,
2718, 2719, 2720, 2721, 2684, 2699, 2703, 2704, 2705, 2706, 2658, 2662, 2679,
2603, 2613,
2617, 2585, 2594, 2595, 2596, 2597, 2598, 2599, 2489, 2497, 2498, 2499, 2500,
2501, 2504,
2505, 2507, 2508, 2510, 2511, 2512, 2513, 2514, 2515, 2519, 2520, 2521, 2522,
2525, 2526,
2527, 2528, 2529, 2530, 2531, 2532, 2534, 2535, 2536, 2537, 2539, 2540, 2541,
2542, 2543,
2544, 2545, 2546, 2547, 2550, 2551, 2552, 2553, 2554, 2558, 2563, 2564, 2572,
2580, 2581,
2472, 2482, 2484, 2408, 2464, 2465, 2466, 2467, 2468, 2339, 2349, 2367, 2370,
2372, 2373,
2374, 2375, 2376, 2377, 2378, 2379, 2380, 2382, 2386, 2387, 2389, 2393, 2394,
2395, 2396,
2397, 2398, 2402, 2403, 2404, 2327, 2333, 2334, 2335, 2305, 2315, 2318, 2260,
2268, 2270,
2271, 2272, 2273, 2274, 2275, 2276, 2277, 2278, 2279, 2280, 2281, 2282, 2283,
2284, 2285,
2286, 2287, 2288, 2289, 2290, 2291, 2292, 2293, 2294, 2295, 2296, 2297, 2298,
2299, 2300,
2301, 2232, 2245, 2246, 2248, 2249, 2250, 2251, 2252, 2254, 2255, 2256, 2210,
2218, 2219,
2221, 2222, 2223, 2227, 2228, 2202, 2204, 2205, 2206, 2164, 2180, 2183, 2187,
2188, 2189,
148
CA 02528853 2005-12-23
2190, 2191, 2192, 2193, 2194, 2195, 2196, 2197 and 2198.
Step e) of the method, namely the detection of the specific amplifioates
indicative of the
methylation status of one or more CpG positions according to SEQ ID NOS:I to
SEQ ID NO:64
is carried out by means of rea2 time detection methods as described above, and
wheroin the
sequtncm of said hybridization probes is selected from the group consisting of
SEQ ID
NOS:1162, 1163, 1201, 1202, 1208, 1211, 1217, 1274, 1288, 1275, 1289, 1299,
1308, 1309,
1341, 1342, 1347, 1351, 1354, 1372, 1423, 1424, 1425, 1427, 1432, 1433, 1434,
1435, 1436,
1437, 1440, 1441, 1442, 1443, 1444, 1445, 1446, 1447, 1370, 1371, 1400, 1450,
1461, 1451,
1462, 1467, 1470, 1473, 1475, 1535, 1536, 1542, 1544, 1569, 1570, 1583, 1590,
1595, 1666,
1704, 1596, 1601, 1603, 1667, 1705, 1722, 1723, 1727, 1735, 1746, 1747, 1768,
176%1773,
1803, 1804, 1807, 1810, 1832, 1833, 1836, 1867, 1871, 1901, 1902, 1908, 1911,
1924, 1925,
1937, 1938, 1943, 1946, 1949, 1956, 1957, 1978, 1979, 1986, 1992, 1998, 2001,
2003, 2010,
2020, 2025, 2029, 2030, 2113, 2163, 2201, 2209, 2231, 2259, 2304, 2326, 2338,
2348, 2351,
2369, 2407,2471, 2488, 2557, 2584, 2602, 2616, 2657; 2661, 2664, 2671, 2683,
2709 and SEQ
ID NO: 2724.
For each genomic sequence listed, the following oligonucteotides as detailed
in the
sequenca listing may be used for a combined Blockingoligonucleotide-RealTime
analysis:
Lightcycler dual probe detection assays:
SEQ ID NO: 41
left Primer.1161,1165,1166, 1167,
right Primer.1160,1168,1169,1170, 1175,
Blocker: 1164, 1171, 1172, 1173, 1174, 1176, 1177,1178,1179,1180,1181, 1182,
1183,1184,1185,1186,1187,1188,1189,1190,1191,1192,1193,1194,1195,1196,1197,
1198,
Detection: 1162,
Anchor: 1163,
SEQ ID NO: 6
left Primer: 1200,1204,1205,1206,1207,1209,1210,1212,1213,1214,1215,1216,
149
osT
'~8~i 'Z8~I '18~I '08~I '6L~i '8L~I 'LL~T '9L~I 'SL~I 'VL~I '~L~I '69~I
uaum{Uoi
I=ox aQ aas
'Vs~I 'IS~I 'Lb~I 'ZV~I :juqouV
'ib~i moAmoQ 0~
'L9~i '99~I 'S9~I '"~i '~9~I 'Z9~I 'I9~I '09ET '69~T '8S~i '~b~i -=Xaoig
'LS~I '9S~t '6~~i :Zmmd 3'R$u
'SS~I '~S~I 'ZS~i 'OS~I '6b~I 'Sb~I '9b~i 'Sb~I 'trV~i 'Ob~I =UM.d 8OI
t~~orlaia~s
sz
'60~T :xotIOUV
'80~I :Qorm;oQ
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'LZ~I 'SZ~T 'bZ~T 'ZZ~T 'iZ~T 'OZ~T '6I~i '8T~T 'LT~i '9i~I 'SI~T 'OT~I
:",3pOIff
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OZ
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corl aQ bas
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'88Zi 't+LZI :UDI
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'40~i '98ZI 'S8Zi 'b8Zi 'ZLZi mumd tqBN
'~0~I 'ZO~I IO~I 'OOEI '86ZI UZI '96ZI 'S6ZI 'V6ZI
UZI UZI 'I6Zi 'L8ZI '~8ZT UZI 'iSZI '08ZT '6LZI UZI 'LLZi '~LZI Mul9d }TSI
tii =oid QT aa5 OI
'LIZI 'IIZI '80ZI 'ZOZI :xpqOUV
'iOZi ~O40%0Q
'69ZT '89Zi 'S9ZT '9SZi 'SSZI 'BtrZI 'Lb'ZI 'OtiZI '6~ZT '8~ZI 'BZZI '~OZI
U03[00IS
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'OLZI 'L9ZI '99ZI 'V9ZI 'f9ZI 'Z9ZI '19ZI '09Zi '65ZI '8SZI 'LSZI 'tlSZI '~SZT
'ZSZT 'TSZI
'OSZI '6*ZI '9tfLT 'StiZI 'MI '~VZI 'ZtrZI 'TVZT 'L~ZI '9~ZT 'S~ZI 'tr~Zi
'~~ZI 'Z~ZI 'i~Zi
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=mudRIP
'RIT.t
~Z-ZT-SOOZ ~588ZSZ0 Fi'J
CA 02528853 2005-12-23
1384, 1385, 1386,1387, 1388, 1389, 1390, 1391, 1392, 1393, 1394,1395, 1396,
1397, 1398,
1399,1401,1402,1403,1404,1405,1406,1407,1408,1409,1410,1411,1412,1413,1414,
1415, 1416,1417, 1418, 1419, 1420,1421,
rsght Primer:1368,1422,1426,1428,1429,1430,1431,1438,1439,
Blocker: 1372,1423,1424, 1425,1427,1432, 1433,1434, 1435, 1436,1437,1440,
1441,1442,1443,1444,1445, 1446,1447,
Detection: 1370,
An+ctyor.1371, 1400,
SEQ ID NO: 9
left Primer: 1449,1453,1454,1460,1464, 1465, 1466,1468,1469,1471,1472,1474,
1476, 1477, 1478, 1479, 1480, 1481, 1482, 1483,1484, 1485,1486, 1487, 1488,
1489, 1490,
1491, 1492, 1493, 1494, 1495, 1496, 1497, 1498, 1499, 1500, 1501, 1502, 1503,
1504, 1505,
1506,1507,1508,1509,1510,
right Primer: 1448, 1459,1511,1512,1513,1520, 1521, 1522, 1523, 1530,
Blocker: 1452,1455,1456,1457, 1458,1463,1514, 1515, 1516, 1517, 1518, 1519,
1524,1525,1526,1527,1528,1529,1531,1532,
Detection:1450,1461,
Anchor.1451, 1462, 1467,1470,1473,1475,
SEQ ID NO: 46
left Primea: 1534,1538, 1539, 1540, 1541,1543,1545,1546,1547, 1548, 1549,
1550,
1551,1552,1553,1554,
right Primer: 1533,
Blocker:1537,1555, 1556, 1557,1558,1559, 1560, 1561,1562,1563, 1564,1565,
1566,
Detection: 1535,
Anchor. 1536,1542,1544,
SEQ ID NO:10
left Primer. 1568, 1572, 1573,
right Primer:1567,1574,1575,1576,1577,1578,1580,1582,1589,
Bloeker:1571, 1579, 1581,1584,1585, 1586,1587,1588,1591, 1592,
Detection: 1569,
151
CA 02528853 2005-12-23
Amhor. 1570,1583,1590,
SEQ ID NO: 12
left Primer.1594,1598,1599,1600,1602,1604,1605,1606, 1607, 1665, 1669, 1670,
1671,1672,1673,1674,1675,1676,1677,1678,1679,1680,1681,1682, 1703,1707, 1708,
right Primer. 1593, 1608, 1632,1664,1683, 1702,1709,1710, 1711,1714,
Blocker.1597,1609,1610,1611,1612,1613,1614,1615,1616,1617,1618,1619,
1620, 1621,1622,1623, 1624,1625,1626,1627,1628,1629,1630,1631,1633,1634,1635,
1636,1637,1638,1639,1640,1641,1642,1643,1644,1645,1646,1647,1648,1649,1650,
1651,1652,1653, 1654,1655,1656,1657, 1658, 1659, 1660, 1661, 1662,1663, 1668,
1684,
1685,1686,1687,1688,1689,1690,1691,1692,1693,1694,1695,1696,1697,1698,1699,
1700, 1701, 1706, 1712, 1713, 1715, 1716, 1717, 1718, 1719,
Deteation:1595,1666,1704,
Anchor. 1596, 1601, 1603, 1667, 1705,
SEQ ID NO: 13
left Prinner:1721,1725,1726,1728,1729,1730,1731,1732,1733,1734,1736,1737,
right Primer. 1720, 1738, 1739, 1740, 1742, 1743,
Biocker:1724, 1741,
Detection:1722,
.Anchor. 1723,1727,173 5,
SEQ ID NO:3
left Primer: 1745, 1749, 1750, 1751, 1752, 1753, 1754, 1755, 1758,
25. right Primer.1744, 1756,1757,
Blocker: 1748, 1759,1760,1761, 1762,1763, 1764,1765,
Detection: 1746,
Anchor: 1747,
SEQ ID NO: 18
left 1'rimer. 1767, 1771, 1772, 1774, 1775, 1776, 1777, 1778,
right Primer. 1766, 1779, 1780, 1781, 1783, 1784, 1787, 1791, 1793, 1794,
Blocker: 1770, 1782, 1785, 1786, 1788, 1789, 1790, 1792, 1795, 1796, 1797,
1798,
1799,1800,
152
CA 02528853 2005-12-23
Debection: 1768,
Anchor:1769,1773,
SEQIDNO:17
left Primesc:1802,1806,1808,1809,1811,1812,1813,1814,
rigbt Primer. 1801, 1815, 1816, 1817, 1818, 1820, 1822,
Blocker.1805,1819,1821,1823,1824,1825,1826,1827,1828,1829,
Detection: 1803,
Anchor. 1804,1807,1810,
SEQ ID NO: 21
left Prinaer=.1831,1835,1837,1838,1839,1840,1841,1842,1843,1844,1845,1846,
1847,1848, 1849, 1850,1851, 1852,1853,1854,1855,1856,1857,1858, 1859,1860,
1861,
1862,1863,1864, 1865, 1866,1868,1869,1870,1872,1873,1874, 1875,1876, 1877,
1878,
1879,1880,1881, 1882,1883,1884,1887,1889,1890, 1891, 1895,
right Primer: 1830,1885,1886,1888,1892,1893,1994,1897,1998,
Blocker. 1$34,1896,
Detection: 1832,
Anchor. 1833,1836,1867,1871,
SEQ ID NO: 35
left Primer: 1900, 1904, 1905, 1906,
right Primer: 1899, 1907, 1909, 1910, 1912, 1913, 1914, 1915, 1916,
1918,1919,1920;
Blocker: 1903, 1917,1921,
Detection: 1901,
Anchor: 1902, 1908, 1911,
SEQ ID NO: 25
left Primer: 1923,
right Primer: 1922,1927,1928,1929,
Bloaker: 1926, 1930,1931,1932, 1933, 1934,
Detection: 1924,
Anchor. 1925,
153
CA 02528853 2005-12-23
SEQ ID NO: 26
left Primer. 1936,1940,1941,1942,1944,1945, 1947, 1948,1950,1951, 1952,
right Primer:1935,1953,1954,1955,1958,1959,1960,1961, 1962, 1963, 1964, 1965,
1967,1968,1969,1970,1971, 1972,
Blocker: 1939, 1966, 1973, 1974, 1975,
Detection: 1937,
Anchor: 1938,1943,1946,1949,1956,1957,
SEQ ID NO: 38
left Primer:1977,1981,1982,1983,1984,1985,1987,1988, 1989, 1990, 1991, 1993,
1994, 1995, 1996, 1997, 1999, 2000, 2002, 2004, 2005, 2006, 2007, 2008, 2009,
2011, 2012,
right Primer. 1976, 2013, 2014, 2015, 2016, 2017, 2019, 2021, 2024,
Blocker. 1980, 2018, 2022, 2023, 2026,
Detection: 1978,
Anchor: 1979,1986,1992,1998,2001, 2003, 2010, 2020, 2025,
SEQ m NO: 27
left 1'rimer. 2028, 2032, 2033, 2034, 203.5, 2036, 2037, 2038, 2039, 2040,
2041, 2042,
2043, 2044, 2045, 2046, 2047, 2048, 2049, 2050, 2051, 2052, 2053, 2054, 2055,
2056, 2057,
2058, 2059, 2060, 2061, 2062, 2063, 2064, 2065, 2066, 2067, 2068, 2069, 2070,
2071, 2072,
2073, 2074, 2075, 2076, 2077,
right Primer: 2027, 2078, 2079, 2080, 2081, 2083, 2084, 2085, 2086, 2088,
2090, 2091,
2092, 2093, 2098, 2100, 2101, 2102, 2103, 2104, 2105, 2106, 2108, 2109, 2110,
Blocker: 2031, 2082, 2087, 2089, 2094, 2095, 2096, 2097, 2099, 2107,
Detection: 2029,
Anchor. 2030,
Taqman Real-time single probe detection assays:
SEQ ID NO: 41
left Primer: 2112, 2115, 2116, 2117,
right I'rimer. 2111, 2118, 2120, 2121, 2122, 2123, 2126, 2128, 2129, 2130,
2131, 2134,
2136, 2145,
Blocker: 2114, 2119, 2124, 2125, 2127, 2132, 2133, 2135, 2137, 2138, 2139,
2140,
2141, 2142, 2143, 2144, 2146, 2147, 2148, 2149, 2150, 2151, 2152, 2153, 2154,
2155, 2156,
154
CA 02528853 2005-12-23
2157,2158,2159,2160,
Detection: 2113,
SEQID NO: 6
left Primer. 2162, 2165, 2166, 2167, 2168, 2169, 2170, 2171, 2172, 2173,
right Primer. 2161, 2174, 2175, 2176, 2177, 2178, 2179, 2181, 2182, 2184,
2185, 2186,
Blockor. 2164, 2180, 2183, 2187, 2188, 2189, 2190; 2191, 2192, 2193, 2194,
2195,
2196, 2197, 2198,
Detection: 2163,
SEQIDNO:8
left Primer. 2199, 2203,
right Primer. 2200,
Blocker: 2202, 2204, 2205, 2206,
Detection: 2201,
SEQ ID NO: 14
left Primer: 2208, 2211, 2212, 2213, 2214,
right Primer: 2207, 2215, 2216, 2217, 2220, 2224, 2225, 2226,
Blocker: 2210, 2218, 2219, 2221, 2222, 2223, 2227, 2228,
Detection: 2209,
SEQIDNO:9
left Primer: 2230, 2233, 2234, 2235, 2236, 2237, 2238, 2239, 2240, 2241, 2242,
2243,
2244,
right Primer. 2229, 2247, 2253,
Blocker: 2232, 2245, 2246, 2248, 2249, 2250, 2251, 2252, 2254, 2255, 2256,
Detection: 2231,
SEQ ID NO: 46
left Primer. 2257, 2264, 2265, 2266, 2267, 2269,
right Primer. 2258, 2261, 2262, 2263,
Bloakar. 2260, 2268, 2270, 2271, 2272, 2273, 2274, 2275, 2276, 2277, 2278,
2279,
2280, 2281, 2282, 2283, 2284, 2285, 2286, 2287, 2288, 2289, 2290, 2291, 2292,
2293, 2294,
155
CA 02528853 2005-12-23
2295, 2296, 2297, 2298, 2299, 2300, 2301,
Detection: 2259,
SEQ ID NO: 13
left Primer: 2303, 2306, 2307, 2308, 2309, 2310, 2311, 2312, 2313,
right Primer. 2302, 2314, 2316, 2317, 2319, 2320, 2321, 2322, 2323,
Blocker: 2305, 2315, 2318,
Detection: 2304,
SEQ ID NO:18
left Primer. 2325, 2328, 2329, 2330,
right Primer. 2324, 2331, 2332,
Blocker: 2327, 2333, 2334, 2335,
Detection: 2326,
SEQ ID NO: 20
left Primer. 2337, 2340, 2341, 2342, 2343, 2344, 2345, 2346, 2366, 2368, 2371,
2381,
2383, 2384, 2385, 2388, 2390, 2391, 2392, 2399, 2400, 2401,
right Primer: 2336, 2347, 2350, 2352, 2353, 2354, 2355, 2356, 2357, 2358,
2359, 2360,
2361, 2362, 2363, 2364, 2365,
Blocker. 2339, 2349, 2367, 2370, 2372, 2373, 2374, 2375, 23'16, 2377, 2378,
2379,
2380, 2382, 2386, 2387, 2389, 2393, 2394, 2395, 2396, 2397, 2398, 2402, 2403,
2404,
Detection: 2338, 2348, 2351, 2369,
SEQ ID NO: 48
left Primer: 2406, 2409, 2410, 2411, 2412, 2413, 2414, 2415, 2416, 2417, 2418,
2419,
2420, 2421, 2422, 2423, 2424, 2425, 2426, 2427, 2428, 2429, 2430, 2431, 2432,
2433, 2434,
2435, 2436, 2437, 2438, 2439, 2440, 2441, 2442, 2443, 2444, 2445, 2446, 2447,
2448, 2449,
2450, 2451, 2452, 2453, 2454, 2455, 2456, 2457, 2458, 2459, 2460, 2461, 2462,
2463,
right Primer. 2405,
Blocker: 2408, 2464, 2465, 2466, 2467, 2468,
Detection: 2407,
SEQ ID NO: 22
156
CA 02528853 2005-12-23
left Primer: 2469, 2483, 2485,
right Primer: 2470, 2473, 2474, 2475, 2476, 2477, 2478, 2479, 2480, 2481,
Blocker. 2472, 2482, 2484,
Detection: 2471,
SEQ ID NO: 19
left Primer: 2486, 2494, 2495, 2496, 2502, 2503, 2506, 2509, 2516, 2517, 2518,
2523,
2524, 2533, 2538, 2548, 2549, 2556,
right Primer: 2487, 2490, 2491, 2492, 2493, 2555, 2559, 2560, 2561, 2562,
2565, 2566,
2567, 2568, 2569, 2570, 2571, 2573, 2574, 2575, 2576, 2577, 2578, 2579,
Blocker: 2489, 2497, 2498, 2499, 2500, 2501, 2504, 2505, 2507, 2508, 2510,
2511,
2512, 2513, 2514, 2515, 2519, 2520, 2521, 2522, 2525, 2526, 2527, 2528, 2529,
2530, 2531,
2532, 2534, 2535, 2536, 2537, 2539, 2540, 2541,
2542,2543,2544,2545,2546,2547,2550,
2551, 2552, 2553, 2554, 2558, 2563, 2564, 2572, 2580, 2581,
Detection: 2488,2557,
SEQ ID NO: 17
left Primer. 2583, 2586, 2587, 2588, 2589, 2590, 2591,
right Primer: 2582, 2592, 2593,
Blocker. 2585, 2594, 2595, 2596, 2597, 2598, 2599,
Detection: 2584,
SEQ ID NO: 21
left Primer: 2600, 2612, 2615, 2618, 2619, 2620, 2621, 2622, 2623, 2624, 2625,
2626,
2627, 2628, 2629, 2630, 2631, 2632, 2633, 2634, 2635, 2636, 2637, 2638, 2639,
2640, 2641,
2642, 2643, 2644, 2645, 2646, 2647,
right Primer. 2601, 2604, 2605, 2606, 2607, 2608, 2609, 2610, 2611, 2614,
2648, 2649,
2650, 2651, 2652, 2653, 2654,
Blocker: 2603, 2613, 2617,
Detection: 2602, 2616,
SEQ ID NO: 36
left Primer: 2655, 2659, 2676, 2677, 2678, 2680,
right Primer: 2656, 2660, 2663, 2665, 2666, 2667, 2668, 2669, 2670, 2672,
2673, 2674,
157
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CA 02528853 2005-12-23
acids have been extracted, the genomic double-stranded DNA is used in the
analysis.
In a preferred embodiment, the DNA may be cleaved prior to the treatment, and
this may
be by any means standard in the state of the art, in particular with
methylation-sensitive
restriction endonucleases.
In the third step, the DNA is then digested with one or more methylation
sensitive
restriction enzymes. The digestion is carried out such that hydrolysis of the
DNA at the
restriction site is informative of the methylation status of a specific CpG
dinucleotide.
In the fourth step, which is optional but a preferred embodiment, the
restriction
fragments are amplified. This is preferably carried out using a polyrnerase
chain reaction, and
said amplificates may carry suitable detectable labels as discussed above,
namely fluorophore
labels, radionuclides and mass labels.
In the f{fdh step the amplificates are detected. The detection may be by any
means
standard in the art, for example, but not limited to, gel electrophoresis
analysis, hybridization
analysis, incorporation of detectable tags within the PCR products, DNA array
analysis, MALDI
or ESI analysis.
Subsequent to the determination of the meWylation state of the genomic nucleic
acids the
presence, absence or subclass of colorectal cell proliferative disorder is
deduced based upon the
methylation state of at least one CpG dinucleotide sequence of SEQ ID NOS:1 to
SEQ II)
NO:64 , or an average, or a value reflecting an average methylation state of a
phuality of CpG
dinucleotide sequences of SEQ lD NOS:1 to SEQ ID NO:64. In an alternative
embodiment the
presence or absence of an aerodigestive cell proliferative disorder selected
from the group
consisting is deduced based upon the methylation state of at least one CpG
dinucleotide
sequence of SEQ II7 NOS:20, 41, 35, 3, 39, 33 , or an average, or a value
reflecting an average
methylation state of a plurality of CpG dinucleotide sequences of SEQ Il)
NOS:20, 41, 35, 3, 39,
33.
Diaznostig Assavs for colorectal ceR Proliferative disorders
The present invention enables diagnosis of events which are disadvantageous to
patients
or individuals in which important genetic andlor epigenetic parameters within
one or more of
159
CA 02528853 2005-12-23
SEQ ID NOS:l to SEQ ID NO:64 may be used as markers. Said parameters obtained
by means
of the present invention may be compared to another set of genetic and/or
epigenetic parameters,
the differences serving as the basis for a diagnosis and/or prognosis of
events which are
disadvantageous to patients or individuals.
More specifically the present invention enables the screening of at-risk
populations for
the early detection of colorectal carcinomas. Furthermore, specific
embodiments of the present
inva-tion enable the debection of aerodigestive cancers and thus are
additionatly suitable for this
purpose.Further embodiments of the method may also be used as atternatives to
cytological
screening for the differentation of colorectal carcinomas from other colon
cell proliferative
disorders, e.g. colon polyps.
Specifi.cally, the present invention provides for diagnostic cancer assays
based on
measurement of differential methylation of one or more CpG dinncleotide
sequences of SEQ ID
NOS:1 to SBQ ID NO:64, or of subregions thereof that comprise such a CpG
dinucleotide
sequence. 'i'ypically, such assays involve obtaining a tissue sample from a
test tissue,
performing an assay to measure the methylation status of at least one of one
or more CpG ;
dinucleotide sequences of SEQ ID NOS:1 to SEQ ID NO:64 derived from the tissue
sample,
relative to a control sample, or a known standard and making a diagnosis or
prognosis based
thereon.
In particular preferred embodiments, inventive oligomers are used to assess
the CpG
dinucleotide methylation status, such as those based on SEQ ID NOS:1 to SEQ ID
NO:535, or
arrays thereof, as well as in ltits based tlsmon and useful for the diagnosis
and/or prognosis of
colorectat cell proliferative disorders.
Kiu
Moreover, an additional aspect of the present invention is a kit comprising,
for example:
a bisulflte-containing reagent; a set of primer oligonucleotides containing at
least two
ofigonucleotides whose sequences in each case correspond, are complementary,
or hybridize
under stringent or highly stringent conditions to a 16-base long segment of
the sequences SEQ
ID NOs:I to SEQ ID NO:535; oligonucleotides and/or FNA-oligomers; as well as
instructaons
160
CA 02528853 2005-12-23
for carrying out and evaluating the described method. In a fiuther preferred
embodiment, said
kit may further comprise standard reagents for perfonning a CpG position-
specific methylation
analysis, wherein said analysis comprises one or more of the following
techniques: MS-SNuPE,
MSP, MethyLight TM, HeavyMethylTM , COBRA, and nucleic acid sequencing.
However, a kit
along the lines of the present invention can also contain only part of the
aforementioned components.
While the present invention has been described with specificity in accordance
with eerhain of its
preferred embodiments, the following examples serve only to illustrate the
invention and are not intended
to limit the iavention within the principles and scope of the broadest
interpretations and equivalent
configurations thereof.
EXAMPLES
Samples were received either as frozen tissue or extracted genomic DNA. DNA
samples
were extracted using lysis buffer from Qiagen and the Roche magnetic
separation kit for
genomic DNA isolation. DNA samples were also extracted using Qiagen Genomic
Tip-100
cohunns, as well as the MagnaPure device and Roche reagents. All samples were
quantitated
using speckrophotometric or fluorometric techniques and on agarose gels for a
subset of samples.
Bisulfite treatmentand mPCR
Total genomic DNA of all samples was bisulfite treat.ed converting
unmethylated
cytosines to uracil. Methylated cytosines remained conserved. Bisulfite
treatment was performed
with minor modifications aceording to the protocol described in Olek et al.
(1996). In order to
avoid processing all samples with the same biological background together
resulting in a
potential process-bias in the data later on, the samples were randomly grouped
into processing
batches. For bisulfite treatment we created batches of 50 samples randomized
for sex, diagnosis,
and tissue. Per DNA sample two independent bisulfite reactions were performed.
After
bisulfitation 10 ng of each DNA sample was used in subsequent mPCR reactions
containing 6-8
primer pairs.
Each reaction contained the following:
0.4 mM each dNTPS
1 Unit Taq Polymerase
161
CA 02528853 2005-12-23
2.5 l PCR buffer
3.5 mM MgC12
80 nM Primerset (12-16 primers)
11.25 ng DNA (bisulfite treated)
Further detaik of the primers are shown in TABLE 1.
Forty cycles were carried out as follows: Denaturation at 95 C for 15 min,
followed by
annealing at 55 C for 45 sec., primer elongation at 65 C for 2 min. A final
elongation at 65 C
was caYried out for 10
1.1.2 vbridization
All PCR products frarn each individual sample were the,n hybridised to glass
slides
cariying a pair of immobilised oligDnucleotides for each CpG position under
anatysis. Each of
these detecfiion oligonu.cleotides was designed to hybridise to the bisulphite
conveated sequence
around one CpG site which was either originally unmethylatsd (TG) or
snethylated (CG). See
Table 2 for further details of all hybridisation oligonucleotides used (both
informative and non-
informative.) Hybridisation conditions were selected to allow the detection of
the single
nucleotide differences between the TG and CO variants.
5 l volume of each multiplex PCR product was diluted in 10 x Ssarc buffer (10
x
Ssarc:230 ml 20 x SSC, 180 ml sodium lauroyl sarcosinate solution 20%, dilute
to 1000 ml with
dH2O). The reaction mixture was then hybridised to the detection
oligonucleotides as follows.
Denaturation at 95 C, cooling down to 10 C, hybridisation at 42 C overnight
followed by
washing with 10 x Ssarc and dH2O at 42 C.
Further details of the hybridisation oligonucleotides are shown in TABLE 2.
Fluorescent signals from each hybridised oligonucleotide were detected using
genepix
scanner and software. Ratios for the two signals (from the CG oligonucleotide
and the TG
oligonucleotide used to analyse each CpG position) were ealculated based on
comparison of
intensity of the fluorescent signals.
The s amples w ere p rocessed i n b atches o f 8 0 s amples randomized for se
x, diagnosis,
tissue, and bisalphite batch For each bisulfite treated DNA sample 2
hybridizations were
162
CA 02528853 2005-12-23
perFormed. This means that for each sample a total nzunber of 4 chips were
processed.
Data analysis methods
Analysis of the chip data:
From raw hybridization intensities to methylation ratios;
The log methylation ratio (log(CG/TG)) at each CpG position is determined
aceording to
a standardized preprocessing pipeline that includes the following steps:
For each spot the median background pixel intensity is subtracted from the
median
foreground pixel intensity (this gives a good estimate of background corrected
hybridization
intensities):
For both CG and TG detection oligonucleotides of each CpG position the
background
corrected median of the 4 redundant spot intensities is taken;
For each chip and each CpG position the log(CG/TG) ratio is calculated;
For each sample the median of log(CG/TG) intensities over the redundant chip
repetitions is taken.
This ratio has the property that the hybridization noise has approximately
constant
variance over the fall range of possible methylation rates (Huber et al.,
2002).
]'~iindpe l Cannponent Analvsis
The principle component analysis (PCA) projects measurement vectors.(e.g. chip
data,
methylation profiles on several CpGs ete.) onto a new coordinate system. The
new coordanate
axes are referred to as principal components. The f~r' st principal component
spans the directaon
of the largest variance of the data. Subseqaent components are ordered by
decreasing variance
and are orthogonal to each other. Difffereat CpG positions contribute with
different weights to
the extension of the data cloud along different components. PCA is an
unsupervised technique,
Le., it does not take into account the labels of the data points (for fiuther
details see e.g. Ripley
(1996)).
PCA is typically used to prnject high dimensional data (in our case
methylation-azray
data) onto lower dimensional subspaces in order to visualize or extract
features with high
163
CA 02528853 2005-12-23
variance from the data. In the present report we use 2 dimensional projections
for statistical
quality control of the data. We investigate the effect of different process
parameters on the chip
data and exclude that changing process parameters cause Iarge alterations in
the measurement
vahies.
A robust version of PCA is used to detect single outlier chips and exclude
them from
further analysis (Model et a1., 2002).
Hypothesis testinug
The main task is to identify markers that show significant differences in the
average
degree of inethylation between two classes. A signifcant difference is
detected when the
nullhypothesis that the average methylation of the two classes is identical
can be rejected with
p<0.05. Because we apply this test to a whole set of potential markers we have
to correct the p-
values for multiple testing. This wag done by applying the False Discovery
Rate (FDR) method
(Dudoit et aL, 2002).
For testing the null hypothesis that the methylation levels in the twa classes
are identical
we u sed the I ikelihood ratio t est for 1 ogistic r egression models
(Venables and R ipley, 2 002).
The logistic regression model for. a single marker is a linear combination of
methylation
measurements from all CpG positions in the respective genomic region o f
interest (ROI). A
significant p-value for a marker means that this ROI has some systematic
correlation to the
question of interest as given b~ the two classes. However, at least formally
it makes no
statement about the actual predictive power of the marker.
Class prediction b~p~ Iearning
In order to give a reliable estimate of how we1l the CpG ensemble of a
selected marker
can differentiate between different tissue classes we can determine its
prediction accuracy by
classification. For that purpose we calculate a methylation profile based
prediction function
using a certain set of tissue samples with their class label. This step is
called training and it
exploits the prior knowledge represented by the data labels. The prediction
accuracy of that
fnnction is then tested by cross-validation or on a set of independent
samples. As a method of
164
CA 02528853 2005-12-23
choice, we use the support vector mschine (SVM) algorithm (Duda (2001),
C'bristiannini
(2000)) to learn the prediction function. If not stated otherwise, for this
report the risk
associated with false positive or false negative classifications are set bo be
equal relative to the
respactive class sizes. It follows that the learning algorithm obtains a class
prediction function
with the objective to optimize acmacy on an independent test sample set.
Therefore sensitivity
and specificity of the resulting classifier can be expected to be
approximately equal.
Bstimatim & oerformance of the tissue class p edi'c~~mgs Valida~ol}
With limited sample size the cross-validation method provides an effective and
reliable
estimate for the prediction aecuracy of a disoriminator functi.on and
therefore in addition to the
significance of the markers we provide cross-validation accuracy, saisitivity
and specificity
estimates. For each classification task, the samples were partitioned into 5
groups of
approximately equal size. Then the learning algorithm was trained on 4 of
these 5 sample
groups. The preclictor obtained by this method was then tested on the
remaining group of
independent test samples. The. number of correct positive and negative
classifications was
counted over 5 runs for the leaming algorithm for all posssble choices of the
independent test
group without using any latowledge obtained from the previous runs. Tbis
procedure was
repeated on up to 10 random permutations of the sample set. Note that the
above-described _
cross-validation procedure evaluates accuraoy, sensitivity and specificity
using practically all
possible combinations of trainin.g and independent test sets. It therefore
gives a better estimate
of the prediction performance than simply splitting the samples into one
training sample set and
one independent test set.
Results
Pigures 1, 5, 9, 13, 16, 20, 24, 28 and 32 show ranked matrices of data
obtained
according to Examples 1 and 2 according to CpG methylation differences between
the two
classes of tissues, using an algorithim. The figures are shown in greyscale,
wherein the most
significant CpG positions are at the bottom of the matrix with significanee
decressing towards
the top. Black indicates total methylation at a given CpG position, white
represents no
165
CA 02528853 2005-12-23
methylation at the particnlar position, with degc+ees of methylation
represented in grey, from
light (low proportion of inethylafion) to dark (high proportion of
inethylation). Each row
represents one specific CpG position within a gene and each column shows the
methylation
profile for the different CpGs for one sample. On the right side p values for
the individual CpG
positions are shown. The p values are the probabilities that the observed
distribution occurred
by chance in the data set.
Figure 96 shows a ranked matrices of data obtained according to Exatnples I
and 2
according to CpG methylation differences between the two classea of tissues,
(aerodigestive
cancer on the left and healthy aemdigestive tissues on the right) using an
aigorilthim. The most
significant CpG positions are at the bottom of the matrix with significance
decreasing towards
the top. Red indicates total methylation at a given CpG position, green
represents no
methylation at the particular position.. Each row represents one specific CpG
position within a
gene and each column shows the methylation profile for the different CpGs for
one sample.
Figures 2, 6, 10, 17, 21, 25, 29 and 33 show the uncorrected multivariate
LogReg test
results for each comparison. Each individual genomic region of interest is
represented as a
point, the dotted line represents the cut off point for the 25% false
discovery rate.
Figures 3, 7, 11, 14, 18, 22, 26, 30 and 34 show ranked matrices of data
obtained
according to Examples 1 and 2 of the accuracy of the genewise linear support
vector machine
cross validations between the two classes of tissues, for the best perforauing
markers. The
figures are shown in gmyscale, wherein the most sigrtifcant CpG positions are
at the bottom of
the matrix with significance decreasing towards the top. Black indicates total
methylation at a
given CpG position, white represents no methylation at the particular
position, with degrees of
methylation represented in grey, from light (low proportion of methylation) ta
dark (high
proportion of inethylation). Each row represents one speeific CpG position
withm a gene and
each column shows the methylation profile for the different CpGs for one
sample. On the right
side accuracy values for each individual genomic region of interest are shown.
Figim 4, 8, 12, 15, 18, 23, 27, 31 and 35 shows the accuracy of the genewise
linear
support vector machine cross validation for all genomic regions of each
classification. The
accuracy of each genomic region is represented as black squares, the
specificity as unMled
166
CA 02528853 2005-12-23
diamonds, the sensitivity as unfilled squares. The accuracy as measured on the
X-axis shows ttLe
fraction of corrwtly classified samples.
CologNormal vs. Colorectai Camcer
In the first comparison 102 colorectal careinoma samples were compared to 73
samples
from nornial colon, including colon polyps and colon inflammatory disorders.
Figure 1 shows the multivariate test results of the top 12 performing markers
using the
conservative Bonferroni corrected Loglteg test.
Figure 2 shows the uncorrected multivariate LogReg test results for each
comparison.
Each individual genomic region of interest is represented as a point, the
dotted line represents
the cut off point for the 25% false discovery rate.
Figure 3 shows the accuracy of the top 12 performing markers.
Figure 4 shows the accuracy of the genewise linear support vector maclxine
cross
validation for atl genomic regions o~ each classification.
The following genomic markers were evaluated as being significalrt:
SEQ ID NUS:i-12,15-20, 22, 25-36, 38-49, 51-58, and oomplements thereof.
QthgZTissues vs. Colorectal Caneer
In this classification 73 colorectal carcinoma samples were compared to an
'other tissue'
class consisting of 140 samples from non-colorectal careinomas, peripheral
blood lymphocytes
and otber normal tissues of non-colorectal origin. These markers therefore
enable the detection
of colorectal carcinoma cells in', for exaznple, body fluids such as serum.
Figure 5 shows the multivariate test results of the top 12 performing markers
using the
conservative Bonferroni corrected LogReg test.
Fig= 6 shows the uncortected multivariate L ogReg test results for each
comparison.
Each individual genomic region of interest is represented as a point, the
dotted line represents
the cut off point for the 25% false discovery rate.
Figure 7 shows the accuracy of the top 12 performing markers.
Figure 8 shows the accuracy of the genewise linear support vector machine
cross
167 -
CA 02528853 2005-12-23
validation for all genomic regions of each classification.
The following genomic markers were evaluated as being signi#icant:
SEQ ID NOS:1-23, 26-36, 38-43, 45-49, 51-58 and complements thereof.
Colon Nonnal and Other Tissue vs. Colon Cancer
In this classification 73 colorectal carcinoma samples were compared to 242
colon
norn-al and 'other tissue' samples. The colon normal class consisted of
healthy colon, colon
polyps and inflammatory disorder colon tissue samples, the 'other tissues'
consisted of samples
from non-colorectal carcinomas, peripheral blood lymphocytes and other normal
tissues of non-
colorectal origin.
Figure 9 shows the multivariate test results of the top 12 performing markers
using the
conservative Bonferroni corrected LogReg test.
Figure 10 shows the uncorrected multivariate LogReg test results for each
comparison.
Each individual genomic region of interest is represented as a poin#, the
dotted line represents
the cut off point for the 25% false discovery rate.
Figure 11 shows the accuracy of the top 12 performing markers.
Figure 12 shows the accuracy of the genewise linear support vector machine
cross
validation for all genomic regions of each classification.
The following genomic markers were evaluated as being sigtuficant:
SEQ ID NOS:1-3, 5-13,15-23, 26-36, 38-43, 45-49, 51-58, and complements
thereof.
Polyps vs. Colorectal Cancer
Tn this classification 73 colorectal carcinoma samples were compared to 51
colon polyp
samples.
Figure 13 shows the multivariate test results of the top performing markers
using the
conservative Bonferroni corrected LogReg test.
Figure 14 shows the accuracy of the top 12 performing maticers.
Figure 15 shows the accuracy of the genewise lz.n.ear support vector machine
cross
validation for all genomic regions of each classification.
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The following genomic markers were evaluated as being siguificant:
SEQ ID NOS:11, 25, 27, 38, 40, 45, 53, and complements thereof.
Colon gormal vs. Colorectal cell praliferat~,ve disorder
In this oalssification 124 colorectal cell proliferative disorder samples
(consisting of
colon polyps and colorectal carcinoma) were compared to 51 'normal colon'
samples consisting
of both heaithy colon saxnples and colon tissue of inflammatory disorders.
Figure 16 shows the multivariate test results of the top 12 performing markers
using the
conservative Bonferroni correeted LogReg test.
Fignre 17 shows the uneorrected multivariate LogReg test results for each
comparison.
Each individnal genomic region of interest is represented as a point, the
dotted line represents
the cut off point for the 25% false discovery rate.
Figure 18 shows the accuracy of the top 12 performing markers.
Figure 19 shows the aecuraey of the genewise linear support vector machine
cross
validation for all genomic regions of each classification.
The following genomic markers were evaluated as being significant:
SEQ ID NOS:1-58, and complements thereof.
Colon Normal vs. Colorectal Cancer
In this classification 73 colorectal carcinoma samples were compared to 51
'normal
colon' samples consisting of both healthy colon samples and colon tissue of
inflammatory
disorders.
Figure 20 shows the multivariate test results of the top 12 performing markers
using the
conservative Bonferroni corrected LogReg test.
Figure 21 shows the uncorrected multivariate LogReg test results for each
comparison.
Each individaal genomic region of interest is represented as a point, the
dotted line represents
the cut off point for the 25% false discovery rate.
Figure 22 shows the accuracy of the top 12 performing markers.
Figare 23 shows the accuracy of the genewise linear support vector machine
cross
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CA 02528853 2005-12-23
validation for aii genonzic regions of each classification.
The following genomic markers were evaluated as being significant:
SEQ ID NOS:1-3, 5-23, 25-36,38-49, 51-58, and complements thereof.
Colon Normal and Other Tissues vs. CQlorectal cell õvrgUferative Disordef
In this classification 124 colorectal cell proliferative disorder sainples
(consisting of
colon polyps and colorectal carcinoma) were compared to a class consisting of
'colon normal'
and 'other tissue' samples. The colon nonnal samples consisted of both healthy
colon samples
and colon tissue of inflammatory disorders, the 'other tissue' samples
consisted of samples from
non-colorectal carcinomas, peripheral blood lymphocytes and other normal
tissues of non-
colorectal origin.
Figure 24 shows the multivariate test results of the top 12 performing makers
using the
conservative Bonferroni corrected LogRegtest.
Figure 25 shows the uncorracted multivariate LogReg test results for each
comparison.
Each individual genomic region of interest is represented as a point, the
dotted line represents
the cut off point for the 25% false discovery rate.
Figure 26 shows the accuracy of the top 12 performing markers.
Figure 27 shows the aceuracy of the genewise linear support vector machine
cross
validation for all genomic regions of each classification.
The following genomic markers were evaluated as being significant:
SEQ ID NOS:1-36, 38-43, 45-58, and complements thereo~
Other Tissue vs. Colon Tissue
The following comparison was carried out in order to identify markers capable
of
discerning elevated levels of free floating colon DNA, especially in bodily
fluids as a marker of
tumor progression. In this classification the 'colon tissue' class consisted
of samples from
colorectal carcinoma, colon polyps, colon tissue of inflannnatory disorders
and healthy colon
tissae. The 'other tissue' class consisted of samples from non-eoloreetal
carcinomas, peripheral
blood lymphocytes and other normal tissues of non-colorectal orlgin.
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Figure 28 shows the multivariate test results of the top 12 perfornning
markers using the
conservative Bonferroni corrected LogReg test.
Figure 29 shows the uncorrected multivariate LogReg test results for each
comparison.
Each individual genomic region of interest is represented as a point, the
dotted line represents
the cut off point for the 25% felse discovery rate.
Figure 30 shows the accuracy of the top 12 performing markers.
Figure 31 shows the accuracy of the genewise linear support vector machine
cross
vaiidation for all genomic regions of each classification.
The following genomic markers were evaluated as being significant:
SEQ ID NOS:1-58, and complements thereof.
N=ai Tissue vs. Cell Proliferative Disorder Tissue
In this classification the gene panel was assessed for its ability to
accurately discriminate
cell proliferative disorder samples from both colorectal carcinoma, colon
polyps and non-colon
origin cancers from 'normal tissues', namely healthy colon samples, colon
tissue of
inflammatory disorders, peripheral b lood 1 ymphocytes, o ther n ormal t
issues o f n on-colorectal
origin~
Figure 32 shows the multivariate test resalts of the top 12 performing markers
using the
conservative Bonferroni corrected LogReg test.
Figure 33 shows the uneorrected multivariate LogReg test results for each
comparison.
Each individual genomic region of interest is represented as a point, the
dotted line repre,sents
the cut off point for the 25% false discovery rate.
Figure 34 shows the accuracy of the top 12 performing marlcers.
Figure 35 shows the accuracy of the genewise linear support vector machine
cross
15 validation for all genomic regions of each classification.
The following genomic markers were evaluated as being significant:
SEQ II) NOS: 1-36, 38-43, 45-49, 51-58, and complements tlaereof.
Aerodigestive canc y. hW~,iy aerodiQestive tisgues
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In this classification ft te,rm 'awodig;tive' describes a sanzple set of
tissues consisting
lung, liver, pancreas, bile duct, stomach, and colon. Figure 96 shows a ranked
matrices of data
obtained according to Examples I and 2 according to CpG methylation
differences between the
two classes of tissues, (Cancerous tissues on the left of the matrix were
compared to normal
tissues on the right of the matrix.) using an algorithim. The most significant
CpG positions are
at the bottom of the matrix with significance decreasing towards the top. Red
indicates total
methylation at a given CpG position, green represents no mthylation at the
particular position.
Each row represents one specific CpG position witbin a gene and each column
shows the
methylation profile for the different CpGs for one sample.The following
genomic markers were
considered significant SEQ ID NOS: 20, 41, 35, 3, 39 & 33.
The following examples descnbe the analysis of the methylation status of the
genomic
sequences SEQ ID N0:35, SEQ ID NO:34, SEQ ID N0:39, SEQ ID N0:29 in healthy
and sick
colorectal cell proliferative *disorder samples. The initial link between said
genes and colorectal
cell proliferative disordecs was initially carried by means of hybridisation
analysis as descn'bed
in EXAMPLE 1. The sequences were then selected from the larger set of genes
analysed in said
example, and the correlation between methylation status and colorectal cell
proliferative
disorder states was validated by analysis of samples using other methylation
analysis techniques,
namely the MSP- MethyLight TM and HeavyMethyl T"A MethyLight T"A assays.
Please note that
the term 'MethyLight TM' is used to describe real time PCR analysis of
bisulfite treated DNA
using probes of both the TaAmanT"! (single probe) and Lightcycler TM(dual
probe) technologies.
EXAMPLE 2
Analysis of inethylation within colon cancer using an MSP- MethyLight assay
(SEQ ID
NO 35) DNA was extracted from 33 colon adenocarcinoma samples and 43 colon
normal
adjacent tissues using a Qiagen entraction kit. The DNA fram each sample was
treated using a
bisulfite solution (hydrogen sulfite, disulfite) according to the agarose-bead
method (Olek at al
1996). The hnatment is such that all non methylated cytosines within the
sample are converted
to thymidine. Conversely, 5-methylated cytosines within the sample reenain
unmodified.
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The methylation status was determined with a MSP-MethyLight assay designed for
the
CpG island of interest and a control fragment from the beta actin gene (Eads
et al., 2001). The
CpG island assay covers CpG sites in both the primers and the Taqman style
probe, while the
control gene does not. The control gene is used as a measure of total DNA
concentration, and
the CpG island assay (methylation assay) determines the methyiation levels at
that site.
Methods: The SEQ ID NO 35 gene CpG island assay was performed using the
following
primers and probes:
Forward Primer: CGGAGGGTACGGAGATTACG (SEQ ID NO:14387);
Reverse Prinaer. CGACGACGCGCGAAA (SEQ ID NO: 14388); and
Probe: CGAAACCCTAAATATCCCGAATAACGCCG (SEQ ID NO: 14389).The
corresponding control assay was performed using the following primers and
probes:
Primer: TGGTGATGGAGGAGGTTTAGTAAGT (SEQ ID NO: 14390);
Primer: AACCAATAAAACCTACTCCTCCCTTAA (SEQ ID NO:14391); and Probe:
ACCACCACCCAACACACAATAACAAACACA (SEQ ID NO: 14392)
The reactions were run in triplicate on each DNA sample with the following
assay
conditions:Reaction solution: (900 nM primers; 300 nM probe; 3.5 mM Magnesium
Chloride; 1
unit of taq polymerase; 200 M dNTPs; 7 1 of DNA, in a final reaction volume
of 20'.
l);Cycling conditions: (95 C for 10 minutes; then 50 cycles of: 95 C for 15
seconds; 60 C for I
minute ).
The d ata w as a nalysed u sing a P MR c alculation p reviously d escribed i n
t he l iterature
(Eads et al 2001).Results. The mean PMR for normal samples was 0.15, with a
standard
deviation of 0.18. The mean PMR for tmnour samples was 17.98, with a standard
deviation of
18.18. The overall difference in methylation levels between tumour and normal
samples is
significant in a t-test (p=0.00000312). The results are shown in Figure 36. A
Receiver
Operatnng Characteristic curve (ROC curve) of the assay was also deter,mined A
ROC is a plot
of the true positive rate against the false positive rate for the different
possible cutpoints of a
diagnostic test. It shows the tradeoff between sensitivity and specificity
depending on the
selected cutpoint (any increase in sensitivity will be accompaaied by a
decrease in specificity).
The area under an ROC curve (AUC) is a measure for the accuracy of a
diagnostic test (the
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CA 02528853 2005-12-23
larger the area the better, optimum is 1, a random test would have a ROC curve
lying on the
diagonal with an area of 0.5; for reference: J.P. Egan. Signal Detection
Theory and ROC
Analysis, Academic Press, New York, 1975). The AUC for the MSP-MethylLightr"A-
Assay is:
0.94 (Figirre 37).
EXAMPLE 3
Methylation of SEQ ID NO 35 within colon cancer was analysed using a
HeavyMethyl
MetbyLightTM assay. The same DNA samples were also used to analyse methylation
of the CpG
island with a HeavyMethyl MethyLight"" (or HM MethyLightT") assay, also
referred to as the
HeavyMethyl"" assay. Tlxe methylation status was determined with a HM
MethyLiglat'"' assay
designed for the CpG island of interest and the same control gene assay
described above. The
CpG island assay covers CpG sites in both the bloekers and the TaqmanTM style
probe, while the
control gene does not.
Methods. The CpG island assay (methylation assay) was perfornned using the
following
primers and probes:
Forward Primer. GGTGATTC'rTTTATTGTTATGGTTTG (SEQ ID NO:14393)
Reverse Prinaer: CCCCTCAACCTAAAAACTACAAC (SEQ ID NO:14394)
Foravard Blocker. GTI'ATGGTTTGTGATTTTGTGTGGG (SEQ ID NO: 14395)
Reverse Blocker. AAACTACAACCACTCAAATCAACCCA (SEQ ID NO: 14396)
Probe: AAAATTACGACGACGCCACCCGAAA (SEQ ID NO: 14397)
The reactions were each run in triplicate on each DNA sample with the
following assay
conditions:
Reaction sohdiot:: (400 nM primers; 400 nM probe; IOpM both blockers; 3.5 mM
magnesium chloride; lx ABI Taqman buffer, 1. unit of ABI TaqGold polymerase;
201iM
cIIVTPs; and 70 of DNA, in a final reaction volume of 20 0);
Cycling conditions: (95 C for 10 minutes); (95 C for 15 seconds, 64 C for 1
minute (2
cycles)); (95 C for 15 seconds, 62 C for 1 minute (2 cycles); (95 C for 15
seconds, 60 C for I
minute (2 cycles)); and (95 C for 15 seconds, 58 C for 1 minute, 60 C for 40
seconds (41
cycles)).
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Results. The mean PMR for normal samples was 1.12 with a stavdard deviation of
1.45.
The mean PMR for tumour samples was 38.23 with a standard deviation of 33.22.
The overall
difl'erence in methylation levels between tumour and normal samples is
significant in a t-test
(p~.000000326). The results are shown in Figure 36.
A ROC curve of the assay was also deternuned_ The AUC for the MSP-Methyl-Light-
Assay is 0.91 (Figure 38)
The assay was tested on an additional set of colon samples (25 adenocarcinoma,
33
normals, and 13 adenomas). The results showed a significant difference again
(Figure 39). The
ROC are shown in Figure 40-42.
IO The MSP and HeavyMethyl variants of the MethyLight assay were determined to
be
equivalent for the analysis of methylation in SEQ ID NO:35. Figure 48 shows
the regression
plot of the percentage methylation detected in each sample using the two
methods.
EXAMPLE 4
The SEQ ID NO:35 -HeavyMethyl-MethyLight-assay was also tested against a panel
of
other tissues (Fignre 43). Besides the colon cancer samples only one of the
two breast cancer
tissues were methylated. However, on a panel of 21 additional breast tumours
(different stages),
only one was methylated (Figure 44). So the marker is specific for colon
tumour samples. All
primers, probes, blockers and reaction conditions were identical to those used
in the analysis of
the colon cancer sainples (Example 3).
EXAMPLE 5
Twelve of the colon tissues analysed by real-time PCR also had paired serum
taken
before surgery. We extracted DNA from 1 mi of that serum using a Qiagen
[J1traSensO DNA
extraction k it, b isuifite treated the DNA s ample, and ran the SEQ ID N 0:35
- HeavyMethyl-
MethyLight""-assay on those samples. The control gene did not amplify for
three of the cancer
sernm samples and three of the normal serum samples, so we can conclude that
the sample
preparation did not work in these cases. In the other cases, there was
evidence of higher
methylation in the cancer samples than the normal samples (Figure 45).
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EXAMPLE 6
Analysis of inethylation within colon cancer using a SEQ ID NO:34 -MSk-
Met1Fyl.ightTM Assay. The colon cancer samples described in Example 2 were
also analysed
S using a SEQ ID NO:34 -MSP- MethyLig61T"" Assay with a Taqman style probe.
The sample
preparation was c,arried out as described above (Example 1). The assay was
performed using the
following primers and probes:
Forward Primer. TGGGATTAAGATTTTCGGTTAGTTTC (SEQ ID NO:14398)
Reverse Primer. CACTACAACGCTACGCGACTAAA (SEQ ID NO:14399)
Probe: TCGACGTTACCCAAACGAATCACATAAAAAAC (SEQ ID NO:14400)
The corresponding control assay was performed as described above (EXAMPLE 2)
The reactions were run in triplicate on each DNA sample with the following
assay
conditions:
Reactfon soluiion: (900 nM primers; 300 nM probe; 3.5 tnM magnesium chloride;
1
units of taq polymezsse; 20Q M dNTPs, 5 M blocker; and 7. l of DNA, in a
final reaction
volume of 20 l);
Cyclfng cwnditions: 95 C for 10 minutes; (95 C far 15 seconds, 60 C for 1
minute) 50
cycles
The d ata w as analysed u sing a PMR c alculation p reviously d escn'bed i n
tb.e literature
(Eads et al 2001).
Results. The results are shown in Figure 36. The mean PMR for normal samples
was
3.93, with a standard deviation of 3.57. The mean PMR for tumour samples was
23.06, with a
standard deviation of 20.23 The overall difference in methylation levels
between tumour and
normal samples is significant in a t-test (p=0.000003063). The ROC curve of
the assay is shown
in Figure 46. The AUC is 0.84.
This was further confirmed using a SEQ II} NO:34 -HeavyMethyl MethyLightTM
assay,
using dual Lightcyclee" probes.
Methods. The CpG island assay (methylation assay) was perfnrmed using the
following
primers and ptobes:
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CA 02528853 2005-12-23
Forward Primer. TGGATAGGAGTTGGGATTAAGATTTT (SEQ ID NO:14401)
Reverse Primer. CTTATTACAATTTAAAEIAAAAAAT"TCACTACAA (SEQ ID
NO:14402);
Blocker: AAATTCACTACAACACTACACAACTAAATTCAACATTAC (SEQ ID
NO:14403);
Probe: TT"I"TCGTATTTTTT"TTCGGGTTATTACGTTTT-Fluor (SEQ ID NO: 14404);
Probe: LC640-ATGTGATTCGTT"TGGGTAACGTCGA Phos (SEQ ID NO:14405.
The reactions were each run in triplicate on each DNA sample with the
following assay
oonditions:
Reaction conditions: 500 nM prirners; 10 M blocker; 250 nM probes; 4 mM
Magnesium Chloride
Cycling profile: 95 C denaturation for 10 minutes; 50 cycles: 95 C 10 seconds,
57 C 30
seconds, 72 C 20 seconds
EXAMPLE 7
Analysis of methylation within colon cancer using a SEQ ID NO:29 -MSP-
MethyLight'"" Assay. The colon c ancer samples d escnbed in Example 2 w ere
also analysed
using a SEQ ID NO:29 -MSP- MethyLightTM' Assay with a Taqman style probe. The
sample
preparation was carried out as described above (Example 2). The assay was
performed using the
foilowing primezs and probes:
Forward Primer. TTTTT3 TTTCGGACGTCGTTG (SEQ ID NO: 14406)
Reverse Primer: CCTCTACATACGCCGCGAAT (SEQ ID NO: 14407)
Probe: AATTACCGAAAACATCGACCGA (SEQ ID NO:14408)
The reactions were run in triplicate on each DNA sample with the following
assay conditions:
Reaction solution: (900 nM primers; 300 nM probe; 3.5 mM magnesium chloride; 1
units of taq polymerase; 20Q pM (NTPs, 5 M blocker; and '1 l of DNA, in a
final reaction
volume of 20 l);
Cycling conditions: 95 C for 10 minutes; (95 C for 15 seconds, 60 C for 1
minute) 50
cycles
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The corresponding control assay was performed as desamW above (EXAMPLE 2).
The d ata w as analysed u sing a P MR c aiculation previously described in the
1 iterature
(Eads et a12001).
Results. The resalts are shown in Figure 36. T'ae mean PMR for noimal samples
was
3.04, with a standard deviation of 4.21. The mean PMR for tumour samples was
21.38, with a
standard deviation of 24.08 The overaU difference in methylation levels
between tumour and
normal samples is significant in a t-test (p=0.0000101973). The ROC curve of
the assay is
shown in Figure 47. The AUC is 0.80.
This was fiuther confirmed using a SEQ ID N0:29 -HeavyMethyl MethyLightTM
assay
(using dual labeled Lightcycl$rTM probes.
Methods. The CpG island assay (methylation assay) was performed using the
following
primers and probes:
Forward Primer: GTAGGGTTATTGTTTGGGTTAATAAAT (SEQ ID NO: 14409)
Reverse Primer. T CTCCTCTACATAC (SEQ ID
NO:14410)
Blocker AACTCCTCTACATACACCACAAATAA.A,TT (SEQ II) NO:14411)
Probe: CGAAAACATCGACCGAACAACG-Fluor (SEQ II) NO:14412}
Probe: LC640-GTCCGAAAAAAAAAAAACGAACTCC-Phos (SEQ II) NO: 14413)
The reactions wera each run in tripiicate on each DNA sample with the
following assay
conditions:
Rgaction conditions: Forward primer: 600 nM; Reverse primer. 30 nM; Blocker.
10
M; Probes: 500 nM; Taq polymerase: 0.1 Ul l; dNTPs: 0.2mM each; Magnesium
Chloride: 4
mMiBSA: 0.25 mg/ml; Roche buffer with no MgClz: lx
Cyci,ne conditions: 95 C danaturation for 10 minutes; 50 cycles: 95 C for 10
seconds,
57 C for 25 seconds, 72 C for 10 seconds
EXAMPLE 8
Analysis of inethylation within oolon cancer using a SEQ ID NO:29 -MSR
MethyLightT"' Assay. An additional assay for SEQ ID NO:29 was tested on colon
samples. The
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assay was t ested o n t wo s ats o f t issues, e ach w ith 12 c olon a
denocarcinomas a nd 12 n onnal
adj acent tissue sanuples.
The sample preparation was canied out as described above (Example 2). The
assay was
perfozzned using the following primers and probes:
Forward Primer: GGACGT'1"TTTTATCGAAGGCG (SEQ ID NO:14414)
Reverse Primer: GCCACCCAACCGCGA (SEQ ID NO: 14415)
Probe: ACCCGAAATCACGCGCGAAAAA (SEQ ID NO:14416)
The reactions were run in triplicate on each DNA sample with the following
assay
conditions:
Reaction solution: (900 nM primers; 300 nM prnbe; 3.5 mM magttesium chloride;
I
units of taq polymerase; 200, M dNTPs, 5 M blocker; and 7 0 of DNA, in a
final reaction
volame of 20 l);
Cycling conditions: 95 C for 10 minutes; (95 C for 15 seconds, 60 C for 1
minute) 50
cycles. The corresponding control assay was performed as described above
(Example 2)
The data w as analysed u sing a P MR c alculation p reviously described in the
1 iterature
(Eads et al 2001). In both cases, SEQ ID NO:29 was significantly more
rnethylated in the
cancer samples. The ROC curves of the assays are shown in Figures 49 and 50.
The AUC are
0.93 and 1.
EXAMPLE 9
Analysis of methylation within colon cancer using a SEQ ID NO:39 -MSP-
MethyLightTM Assay
The colon cancer samples descn'bed in Example 2 were also analysed using a SEQ
ID
NO:39 -MSP- MethyLights" Assay. The sample preparation was carried out as
described above
(EXAMPLE 2). The assay was performed using the following primers and probes:
Forward Primer: GACGGATTTTTTP!"TAACGTTT'I1TC (SEQ ID NO:14417)
Reverse Primer: AAATAAAATACCACCTCCGCGA (SEQ ID NO: 14418)
Probe: GCTCCTCGCGAAATACTCACCCCG (SEQ ID NO:14419)
The reactions were ran in triplicate on each DNA sample with the following
assay
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conditions:
Reaction solution: (900 nM primers; 300 nM probe; 3.5 mM magnesium chloride; 1
units of taq polymerase; 200, pM dNTPs, 5 M blocker, and 7 t of DNA, in a
final reaction
volume of 20 l);
Cycling conditions: 95 C for 10 minutes; (95 C for 15 seconds, 60 C for 1
minute) 50
cycles.
The conresponding control assay was performed as described above (EXAMPLE 2).
.The data was analysed using a PMR calculation p reviously d esen'bad in the l
iterature
(F=ads et al 2001).
Results. The results are shown in Figura 36. The mean PMR for normal samples
was
2.25, with a standard deviation of 2.42. The mean PMR for tamour samples was
25.67, with a
standard deviation of 17.57 The overall difference in methylation levels
between tumour and
normal samples is significant in a t-test (p-0.00000000118). The ROC curve of
the assay is
shown in Figure 52. The AUC is 0.94
This was further confirmed using a SEQ ID NO:39 -HeavyMethyl MethyLightTM
assay,
usang dual Lightcycler probes using Lightcycler style dual probe teclmology.
Methods. The CpG island assay (methylation assay) was performed using the
following
primers and probes:
Forward Primer: GTTAGTTAGITAATTTTTTAAATAGATTAGTAG (SEQ ID
NO: 14420)
Reverse Primef: CAAAAAAACAAATAAAATACCACCTCC (SEQ ID NO:14421)
Blocker. CCTCCACAAAACTCACTCC'PCACA.AAATAC (SEQ ID NO:14422)
Probe: red640 TTTCGTTTTGTATGGTAGA.TACG('xGGTGA- phosphate (SEQ ID
NO:14423)
Probe: ATTAATGGTTTTATAAGACGGATI"1'TTTTTTAACGT- fluorescine (SEQ ID
NO:14424)
The reactions were each run in triplicate on each DNA sample with the
following assay
conditions:
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CA 02528853 2005-12-23
Reaction conditions: Forward primer=. 600 nM; Reverse primer. 300 nM; Blocker.
10
M; Probes: 500 nM; Taq polymerase: 0.1 Ul l; dNTPs: 0.2 mM each; Magnesium
Chloride: 4
mM; BSA: 0.25 mg/rnl; Roche buffer with no MgC12:1x
CLc-hLng conditions: 95 C denahiration for 10 minutes; 50 cycles: 95 C for 10
seconds,
57 C for 25 seconds, 72 C fer 10 seconas,
Fxper'm;õe t 10: Stool analysis of meftlation markers
The following experiments were run in order to establish the feasibility of
inethylation
markers (for the detection of colon cell proliferative disorders) from DNA
derived from stool
samples. Accordingly, a series of samples were prepared each comprising either
stool only
(control) or stool with the addition of a controlled amount of cells from the
colon cancer cell line
SW 620 (25,000 cell equivalent -0.15 ug) the prepared samples were then
frozen. Three
different stool ss4nples wear, used. As a positive control a further sample
was prepared
comprisingperipheral blood lymphocytes (PBLs) and a controlled amount of cells
from the
colon cancer cell line.
The samples were as follows:
Class 1: Stool sample No. I
Class 2: Stool sample No. 1+ 0.15 ug
Class 3: Stool sample No. 2
Class 4: Stool sample No. 2 + 0.15 ug
Class 5: Stool sample No. 3
C]ass 6: Stool sample No. 3+ 0.15 ug
Class T: PBS + 25,000 cell equivalents (0.15 ug)
Class 8: H20
Multiple samples were prepared in each class. 300 ng of stool DNA was present
in each sample.
Each sample was then analysed for methylation using an MSP TaqMan assay for
the gene
EPHB65 as follows:
Primer Forward: T'I7'T1TITTCGGACGTCGTTG (SEQ ID NO: 14455)
Primer Reverse: CCTCTACATACGCCGCGAAT (SEQ ID NO:14456)
Probe: AATTACCGAA.AACATCGACCGA (SEQ ID NO:14457)
Cyocling conditions were: The reaction coiiditions for TaqMan:
181
CA 02528853 2005-12-23
Initial denaturation 95oC 10min-
Denatiuation 95oC 15sec
Armealing/ Extension 6OoC 60sec
50 cycles
Amplification curves of the PCR reaction were according to figures 52 to 58.
Class 1: Figure 52
Class 2: Figure 53
Class 3: Figure 54
Class 4: Figvre 55
Class 5: Figure 56
Class 6: Not applicable (see below)
Class 7: Figure 57
Class 8: Figure 58
The X-axis shows the cycle number of the polymerase chain reaction, the Y-axis
shows
the delta Rn, which is the fluorescence intensity over background, the
horizontal line (A)
represents the threshold level of the calibration curve (B), if the curve
reaches above (A)
methylation has been detected.
As can be seen, methylated colon oancer DNA could be detected in classes 2 &
5. Class
6 was not analysed as methylated DNA was detected in the stool sample (class
5).
In order to detennune the absolute sensitivity of inethylated DNA detection in
stool
samples stool sample No. 2 was spiked with varying amounts of SW 620 DNA. Five
classes of
samples were analysed using the MSP assays above:
= Class B:25,000 cell equivalents in 250 mg stool
= Class C:2,500 cell equivalents in 250 mg stool
= Class D:250 cell equivalents in 250 mg stool
= Class E:25 cell equivalents in 250 mg stool
= Class F:0 cell equivalents in 250 mg stool
The amplification curves can be seen in figure 59. The X-axis shows the cycle
number of the
polymerase chain reaction, the Y-axis shows the delta Rn, which is the
fluorescence intensity
over backgi+ound, the horizontal line (A) reparestnts the threshold level of
the calibmtion curves
(B-F), if the curves reach above (A) methylation has been detected.Curves B,
C, D; E; & F are
the amplification curves of classes B, C, D, E& F respectively. Thus it can be
seen that
methylated DNA can be detected in stool samples at levels of 25 cells per 250
mg stool.
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Example 11:
Real time analysis of inethylatian sensitlve re.striction.
DNA was prepared from from ten colon tumor samples and ten normal adjacent
tissue
samples. DNA isolation was performed using standard methods known to the
expert and
quantified by W measurement.
A digestion reaction was performed with the following reagents:
1 g genomic DNA;
25 U enzyme (10U/ l);
Y+/Tango Buffer
By incubation 37 C.
The following steps were pe,rformed for all samples in parallel. I g of DNA
was
dissolved in 190 1 Y+/Tango Buffer (Fermentas) in 0,2nil PCR tubes. 95 l of
the reaction
solution (half of the overall volume) was pipetted into a ele8n PCR tube as a
control. To the
remaining reagent in the first tube 1,251t1 HpaII and 1,25p1 HirsSI
restriction endunuclease (both
from Fermentas)were added.
To the second tube 2,5 1 of a nzixture of glycerol and water (v:v =1:1) was
added.
After incubating all 40 tubes at 37 C in an EppendorffThermocy+eler far five
hours. Then
another aliquot of restriction enzyme, resp. water-glycerol mix, was added to
each tube and the
incubation was extended for ten hours. After this period of time the
restriction enzymes wem
inactivated by heating at 65 C for 10 minutes.
Subsequently, dilutions of each tube were assembled in 96we11 plates by
transfetring
20 1 from each tube into the plates and adding 80 1 water.
FinaUy, both the restriction product and the control of each sample were
analyzed by
Quantitative Real Time PCR. In tWs example 121i1 of a SybrGreen containing PCR
mastermix
containing one of the primers according to table 4 was pipetted into a 96we11
Taqman plate
followed by 81t1 of the eorresponding dilutions of zestricted sample and blank
(each in
duplicate).
Real Time PCR was performed on a ABI 7700 instrument applying the following
183
CA 02528853 2005-12-23
temperatare profile:15 minutes at 95 C; 50 cycles of:15 seconds at 95 C, 45
seconds at 65 C,
75 seconds at 72 C;15 seconds at 95 C followed by 50 C for one hour.
Using the described primers and conditions amplification is observed only for
methylated copies of the analysed sequences (according to table 4) when
methylation sensitive
restriction has been performed prior to PCR, since unnnethylated copies are
digested during this
step. The amount of inethylated DNA after restriction is normatized against
the total amount of
DNA present before the restrtction as obtained by Real Time quimtitation of
the blank of the
same sample. The equation
,} ect=Ctb,,* -CtõM',,W,
represents a messare for the relative amount of unmethylated DNA present in a
DNA sample. A
value of zero means that the DNA is completely methylated. A value of 0.5
corresporxis to
approximately 50% methylation in case of an optimised PCR reactions whereas
larger values
correspond to low methylation levels. The relative amount of methylated
sequence present in a
sample would be given by.
(2) M - F- o
where E is the PCR efficiency. The PCR efficiency is easily obtained by
methods known to the
expert.
Table 4
esul
Gene Forward primer Reverse Prinaer Figure
SEQ 60
ID GACGGGGGTTCTGGACTTAC CCAGTCCCTGGATGTTGCT
NO: 8 (SEQ ID NO: 14458) (SEQ ID NO:14459)
SEQ 61
ID GCTAAGCCGGAGTCACCAAG CGCTGAGACCGAGAAGAAG
NO: 5 (SEQ IIID NO:14460) (SEQ ID NO:14461)
SEQ GCCGACGAGACCAAGGAG ATGAGCAGCGTGAAGACGA 62
184
S8i
AlMuaptooB sgnn ojdures anssti Isiqozro.tq V '.motum suqouap j, lo 1etzrxou
2un
povap u, se
sFxB-x aqi uo pamoaus st aidures Iunp?Aipq ~'3 tL - 09 ~j3 ut unnoqs aa~
s;Insa~
(isvtit:ou cII bas) (ostrot:Ox (a bas) sv :o
.LOO to,>~tot~o~so,to~~~oo o~.La.LOO,LS~r.~as~roo.to ai
IL bas
(6Litt:Ox (abaS) (8Liti-Old Cl baS) Z :O
JJ.I.JWJMDMVJVJD,LVJ .LJ31JJDDd3fAL3Wd.I7DD CU
oL bsis
(LLbbuOx CII bas) (9LbbI :ox Ca bas) ~9 :0
D,L.Loa.LZ.~~omoo,tayDZD .LD.I.300,V.LVrJDJaYMD,LD a[
69 bas
(SLbbI:Ox Cli bas) (bLtrbi:Ox CII bas) b9 :O
DZrJW'J~DW.L,L~DVDWJD3 ,LDSJODOd,L~~OMDa ,LJ CII
89 bas
(~L"vox (a bas) (ztttr=ox cII bas) 9tr :o
JWW:?VJDVD.LiJ.L3JVrJDM rJJ,LJ,LDJWIADd',L,I.I2V3D CC[
L9 baS
(ILbf-i:ON (II b3S) (OLtbI:Qx (II bas) t9 :p
W,LrJO'd003dOJJJJ,Lfl'd ~WD'~J'da DOiJDZDVVJZ,LD QT
99 bas
(69trtr I:ox ca bas) (89bvi:Ox QI bas) i I:O
J,DV000<JDd,LDJ.JJJ'~JW WJ,LJDdfl~'JVJJWflJ,L.-A tII
S9 bas
(99b bl:oN (n bas) zi :o
(L9ttT:ox CII bas) DWOdrJO'JJVJW,LJd'J'JD CQ
tr9 1.ad'OJE?rJ~VJD,LOOD'JW bas
(s9w:Ox ca bas) (miI:ox cabas) z9 :o
.LrJ1LJJ.iaõL007.L~~3V~J,.Ld .L1.,I.flVrJHHDDD,Ld'JDJ.LJD (11
~9 bas
aI ~o
(~9tr6i:ox (n bas) (z9bbl:ox CCI bas) m
~Z-ZT-SOOZ ~588ZSZ0 Fi'J
CA 02528853 2005-12-23
analysed in some assays, these are aanotated with an armw, and are irrelevant
in the context of
the present invention. The Y-axis shows the level of inethylation
('meEhylation rate ') in the
sample. The methylation rate {methylatedl(methylated + unmethylated) are shown
for the
fragments corresponding to the primer pairs in Table 4. Formula (2) above was
used for the
calculation assuming a PCR efficiency of B= 2.
The data demonstrate that for several fragments the majority of the tumor
samples show
significantly higher methylation rates thaa the corresponding nonnal adjacent
tissues.
Example 12
mRNA expression analysis
1. RNA =Mlg
'Total RNA from normal colon (rr-1) and colon cancer (adenocatcinoma; n=1) was
obtained finm Ambion (Huntingdon, United Kingdom). Each RNA sample was derived
from a
single donor. Total cellular RNA was prepared using the ToTALLY RNA Kit
(Ambion,
Huntingdon, United Kingdom) and Dnase-treatment was performed to minimize
contamination.
RNA integrity was assessed 'before and a8er 14 hours incubation at 37 C on an
Agilent
Bioanalyzer (Agilent Technologies, Inc, Palo Alto, CA).
2. Mi.croarray Experiments
Target preparation and labeling, microarray hybridization, washing, and
scanning was
performed according to the manufacturer's protocol (Affymetrix, Inc., Santa
Clara, CA). Briefly,
10 g of total RNA were reversa transcribed using a T7-linked oligo-dT primer,
followed by
second strand synthesis. Biotin labeled cRNA targets were prepared from double
stranded
cDNA using T7 RNA polymerase and biotinylated ribonucleotides (UTP, CTP).
Fragmentation
was performed at 94 C for 35 minutes. 15 pg of the fragmented biotinylated
cRNA were mixed
with MES buffer (2-(N Morpholino)etb.anesulfonic acid) containing 0.5 mg/ml
acetylated
bovine seram albumin and hybridised to Affymetrix GeneChip HG LT133A arrays
(Santa Clara,
CA) for 16 hours at 45 C. Microarrays were stained using streptavidin
phycoerythrin conjugates
(Molecular Probes, Inc., Eugene, OR). For signal amplification biotinylated
anti-streptavidin
antibodies (Vector Laboratories, Burlingame, CA) were used.
Anmys were scanned on an Affymetrix GeneChip Scanner. The expression values
for
each probe set (representing a gene) were calculated from the raw data using
Bioconductor R
packages 0http:/1www.biocondgctor.org, see below).
DataAnaly'g
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CA 02528853 2005-12-23
The raw expression data obtained from the Affymetrix scanner (the so called
cel-files)
were bsckground corrected and nornnalized using the defanlt settings of the
RMA noimalization
procedure (Frizaxry RA, Hobbs B, Collin F, Beazer-Barclay YD, Antonellis KT,
Scherf U, Speed
TP: Exploration, normalization, and summaries of high density oligonucleotide
array probe level
data. Biostatistics. 2003 Apr;4(2):249-64) as implemented in the Bioconductor
R packages,
release 1.4 (Gautier L, Cope L, Bolstad BM, Irizarry RA: affy-analysis of
A8'ynaetrix
GeneCbip data at the probe level. Bioinfonnatics. 2004 Feb 12;20(3):307-150).
The raw data
processing results in signal intensity values for each of the 22.293 probe
sets available on the
Affymetrix GeneChip HG U133A chip.
To identify diffemtiaUy expressed probe sets, the fold change of the
normalized signal
intensifies between colon normal tissue and colon tumour t.issue was
calculated. Genes with a
fold change bigger than 2 (or smaller than 0.5) were identified as
differentially expressed.
Differentially expressed genes are shown in Table 5, as can be seen
hypermetbylation does not
correlate with underexpression in all cases.
Tabk 5
e og-fold old- up/down in
EQ chanp
Affymetxix
beID O:
08944 at 3 -10 2,0 own
09945 s at 2 1,2 2,3
14211 at 54 -1,3 2,5 wn
03140 at 1 -1 8 3,4 wn
EQ 1D
O:
497 at 15 -1,4 2,6 wn
Example 13
TPF.F(TMf3M) methylation analysis in sertun samples.
In the following example 3 different sample sets of serom from colorectal
cancer patients
and healthy colon individuals were analysed using MSP assay '4' and HM assay
'6' of the geae
ThCEFF2. Healthy colon was defined as being verified by colonoscopy as having
no colon
matignancy or polyps present. The assays were run on the LightCycler platform
(Roche
Diagnostics) , using two different versions of the instxument for comparative
purposes, namely
versions '1.2' and '2Ø They are referred to below as 'LC 1.2' and 'LC 2.0'
respectively.
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CA 02528853 2005-12-23
samule sets
Sample set one:
30 colorectal cancers, 30 colonoscopy verified as no colon malignancy or
polyps
Sample set two:
15 colorectal cancers,15 colonoscopy verified as no colon malignaucy or polyps
Sample set three:
30 colorectal cancers, 30 colonoscopy verified as no colon malignancy or
polyps
In each case approximately 1.8 mis of setvm equivalent was analysed per PCR
Reaction conditiQns
Assay '4' was performed as below.
Forward primer: agggcgttttgttggcg (SEQ ID NO:14482)
Reverse Primer: ctacaacctaaacgacgegct (SEQ II? NO:14483)
Taqman Probe: atcggcgttttagcgtgcggg (FAM/BHQ11abelled) (SEQ ID NO:14484)
Ligbtcxcler program:
' tio = 95 10 mins
3 cycles 950 15 sees (20 /sec)
66 45secs (20 /sec) detection
3 cycles 95 15 secs (20 /sec)
64 45secs (20 /sec) detection
50 cycles 95 15 sees (20 /sec)
62 45secs (20 /sec) detection
Rlts
Assay'4
Sample set one:
AUC=0.95 [0.86, 0.99) (95% confidence intervals) LC 2.0
Sample set two:
AUC-0.65 [0.44,0.80] Z.C 2.0
AUC=0.57 [0.37, 0.75] LC 1.2
Sample set thtee:
AUC=0.83 10.71, 0.92] LC 2.0
AUC=0.83 [0.71, 0.921 LC 1.2
Assay '6'
Forward primer: aaaaaaaaaaaactcctctacatac (SEQ ID NO: 14485)
Reverse Primer: ggttattgtttgggttaataaatg (SEQ 1D NO:14486)
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CA 02528853 2005-12-23
Blocker oligonucleotide: acatacaccacaaataaattaccaaaaacatcaaccaa-pho (SEQ ID
NO: 14487)
Probes: ttttttttttcggacgtcgtt-fluo (SEQ ID N0:14488)and red640-
tcggtcgatgttttcggtaa pho (SEQ
ID NO:14489)
ILightCycler 1.2
rograms
activati,on: 95 C I10min
55 cycles: 95 C 10 sec (20 C/s)
56 C 30 sec (20 C/s) detect:ion
72 C 10 sec (20 C/s)
Sample set two:
AUC-0.70 [0S 1,0.85] LC 2.0
Sample set three:
AUC= 0.69 [0.55, 0.801 LC 2.0
AUC= 0.71 [0.S3, 0.83] LC 1.2 (Some samples were lost, approximately 20 cancer
senun
samples, 20 healthy colon senun samples were analysed)
Exampt 14
MSP analylis of the g;enes acoordins to Table 6(below
In the following analysis the methylation status of a selection of the genes
according to
Table 3 were analysed by means of inethylation specific amplification using
the primers
according to Table 6 (below).
The study was run on approximately 140 samples from colon, breast and liver
carcinoma,
normal tissue (peripheral blood lymphocytes, healthy colon and healthy tissue
adjacent to colon
carcinoma), colon polyps and other colon diseases. The positive class
oonsisted of Colon
Cancer and Colon polyps. The negative class was PBL (peripheral blood
lymphocytes), Colon
Nor.mal, Colon-NAT and Coloz-Inflammatory. For AUC values in braokets (see
table 7, below)
Breast-Cancer and Liver-Canc,er were also included in the negative class.
Genonue DNA was analyzed using the MSP technique after bisulfite conversion.
Total
genomic DNA of all samples was bisulfite tceated converting unmethylated
cytosines to uracil.
Methylated cytosines remained conserved. Bisulfite treatment was performed
with minor
modifications according to the protocol descnbed in Olek et al. (1996).
The sequence of interest was then amplified by means of methylation specific
primers, the
ampli.ficate is then detected by means of inethylatian specific Taqman probes.
189
CA 02528853 2005-12-23
Table 6: Oliaonncleotides for MSP Taqan.
8
.~
F ~ o
~..
aa~
=,..
~, ~..
p4~~
SR ~
190
CA 02528853 2005-12-23
s
ma G, V
CAo~~
52 =- =p
'Zi
g
Ix ~.
pH
Reaction Conditions for Taqman PCR program:
Denaturation at 95 C for 10 minutes.
50 cycles: Denaturation at 95 C for 15 seconds.
Annealing at 62 C for 1 minute. Except Assayl 5, which has an annealing
temp of 60 C.
Amplification curves for each assay are shown in figures 72 to 86 (according
to Table 6).
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CA 02528853 2005-12-23
The technical performance of a series of a[ternative assavs was validated by
testing each of the
followingusingVerm DNA as the tenmlate:
Gene SEQ ID NO: 34 (MSP assay)
Primers: cgtacgaacgca.acaaccg (SEQ ID NO: 14535) & gttcggcgttgtttaggc (SEQ ID
NO: 14536)
Probes: gcatcttccaacaccgtcc,cg (SEQ ID NO: 14537) - fluo & LC640-
ctccgcatccttccccgaa-p
(SEQ ID NO: 14538) (Lightcycler probes)
Lightcycler program:
35 10 mins
55 cycles 95 15 secs(20 /sec)
62 45secs (200/sec)detection
72 15 secs(20 /sec)
Gene SEQ ID NO: 28 (MSP assay)
Primers: ccgaacaaacgtaatacgcg (SEQ ID NO: 14539) & acggcgtgaaggagcg (SEQ ID
NO:
14540)
Probes: tacgcgcggtagtcgtgcgc (SEQ ID NO: 14541) (Taqman probe FAMBHQ1
labelled)
Lightcycler program:
95010 mins
55 cycles95 15 secs(20 /sec)
60 45secs (200/sec) detect:ion
Gene SEQ ID NO: 10 (MSP assay)
Primers : cgacaaaatcctcatcgcg (SEQ ID NO: 14542) & gtcgtttagcgttgggaagc (SEQ
ID NO:
14543)
Probes : tacgtacgagtttcgtacgcgtattatttcgtcg (SEQ ID NO: 14544) (Taqman probe
FAMlBHQI
labelled)
Lightcycler program:
95 10 mins
55 cycles 95 15 secs (20 /sec)
62 45secs (20 /sec) detection
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CA 02528853 2005-12-23
Gene SEQ ID NO: 9 (MSP assay)
Primers: acitasaaatcgccgaatog (SEQ ID NO: 14545) &
agttttatagttaattagggtagttgtcgtcg (SEQ ID
NO: 14546)
Probes: tcxtatcatcgctcacgaacg (SEQ ID NO: 14547) - fluo & LC640-
cgacaacgactatccttactattataa-p (SEQ ID NO: 14548) (Lightcxcler probes)
LC program:
95 10 mine
55 cycles 95 15 secg (200/eec)
60 30 secs (20 /sec) detection
72 15 secs (20 /sec)
Gene SEQ ID NO: 17 (IicavyMethylm assay)
primer:
Ggtgttgtttat#itagagagtt (SEQ ID NO : 14549)
Cctaacccctatcctoc (SEQ ID NO : 14550)
, Iocker:
ccttoaccaccttcccaacactacaacac-phho (SEQ ID NO: 14551)
probes:
Itcggcgtcggtcgttsgg-fluo (SE~ ID NO: 14552)
jred640-cgcgggagtagttagatgcgtc pho (SEQ ID NO: 14553)
ightCycler program:
activati : 95 C 10min
5 ies: 95 C 10 sec (20 Cls)
56 C 30 sec (20 C/s)
72 C 10 sec (20 C/s)
' ony-c 95 C 10sec 0
40 C 10sec 20
70 C Osec 0,1
1~ 40 C 5 sec
193
CA 02528853 2005-12-23
Gene SEQ II) NO: 7(HeavyMethylT" assay)
,Primer :
Tgagagagagagggttgaaa (SEQ ID NO: 14554)
Tct4~aataacaaaatacctccatt (SEQ ID NO: 14555)
;probes:
CGaccCGccaacCGac-fluo ( SEQ ID NO : 14556)
LCred640-CGcCGaaaCGCGctc-p (SEQ ID NO:
14557)
blocker:
Iccattaccaacacaacccaccaaccaa p(SEQ ID NO : 14558)
LightCycler program:
lactivation: 95 C 10min
155 cycles: 95 C 10 sec (20 C/s)
56 C 130 sec (20 C/s) detection
72 C 10 see (20 C/s)
eltin c e: 95 C lOsec 20
40 C l0sec 20
70 C Osec 0,1
o lin : 40 C 5 sec
Gene SEQ ID NO: 8(HeavyMethyP assays)
primer:
~Aaaaaaaaaaaactcetctacatac (SEQ ID NO: 14559)
Ggttattgtttgggttaataaatg (SEQ ID NO : 14560)
blockerr:
Iacatacaccacaaataaattaccaaaaacatcaaccaa pho (SEQ ID
N0 : 14561)
probes:
ttttKttttcggacgtcgtt-fluo (SEQ ID NO: 14562)
,red640-tcggtcgatgttttcggtaa-pho (SEQ ID NO: 14563)
LigLtCycler program:
activa 'on: 95 C 110min
155 cycles: 95 C 110 sec (20 C/s)
194
CA 02528853 2005-12-23
56 C 30 sec 1(20 C/s) detection
72 C~10 sec (20 C/s)
~_..._
eltine curve: 95 C l Osec 20
40 C IOsec ~20
70 C 1Osec 0,1
cool 40 C 5 sec
primer:
Aaaaaaaaaaaactcctctacatac (SEQ ID NO: 14564)
EGgktattgtttgggttaataaatg (sEQ ID NO: 14565)
,blocker:
{ctctacataCAcCCACAaataaattacCAaaaacatC-p (SEQ ID
INO: : 14566)
probes:
CGCGaataaattacCGaaaacatc-fluo (SEQ ID Na: 14567)
LCred640-accgaacaacgacgtcc-p (SEQ ID NO: 14568)
LightCxcier program:
activation: 95 C lOmin
55 c 1: 95 C 10 soc (20 C/s)
56 C 130 sec (20 C/s) detection
72 C 10 sec (20 C/s)
~ pLeltjing curve: 95 C tiOsec 20
40 C 10sec 0
70 C 0sec 10,1
40 C 5 sec
rimer:
gaggtgttagaggagta.gtag (SEQ ID NO : 14569)
ccccctacaacctaaa (SEQ ID NO: 14570)
Iblocker:
Acctaaacaacacactcccacacactaaaacaccaat (SEQ ID NO:
14571)
probes:
Cgagtcggcgcggga-flua (SEQ ID NO: 14572)
195
CA 02528853 2005-12-23
..., .~... ..... , . _. ._., .__._,. .._.
I.ared640-agggcgttttgttggcgatc-p (SEQ YD NO: 14573) ~ ( (
Li~htCycler program:
-activation= 95 C 10min
55 e;g 95 C 10 sec (20 C/s)
58 C 30 sec 1(20 C!s) detection
72 C 10 seo (20 Cls)
cn~urve 95 C Iasec 20
40 C 10seo 20
70 C Osec 0,1
co i g= 40 C 5 sec
Gene SEQ ID NO: 9(HeavyMethyff assays)
jPrnnGr
ctatcaatatacctaactaattaataac (SEQ ID NO : 14574)
igtaaagtttttaatttttattagtattagtat (SEQ ID NO : 14575)
Blocker '
ctaattaataacacttaaaacacaattatamaccaccatao-pho (S8~ ID
O : 14576)
be:
~gdtatgtcgattatagttacgttagttt-pho (SEQ
ID NO: 14577)
gtgaacggtttcgbCgggag fluo ( SEQ ID NO : 14578)
ightCycler program:
activation: 95 C 10min
c es: 95 C 10 sec 20 C!s)
56 C 30 sec (20 C/s) tion
72 C 10 sec (20 C/s)
1' e: 95 C lOsec 0
40 C lOsec 20
70 C sec
[oi
coo ' 40 C 5 sac
196
CA 02528853 2005-12-23
.Pnmer.
jgttttattgtatgatgaattgga (SEQ ID NO: 14579)
caccaaaaaaatattaaccac (SEQ ID NO: 14580)
lBlocker:
Ttaaccacaacacaaataaaaccaaacaaattatccata-
pho (SEQ ID NO: 14581)
Probe:
Gttttttcgcgttcgtcgtttatttt-fluo (SEQ ID
O : 14582)
Red640-ttgggtcgacgagattaaggagg-pho (SEQ ID
O: 14583)
Ligl-tCycler program:
fiv ' n: 95 C l0mirt
c es: 95 C 10 sec (20 C/s)
56 C 30 sec (20 C/s) detection
72 C 10 sec (20 C/s)
curve: 95 C 10sec 20
400C 10sec 3220
70 C sec ,1
.SQEhLnX J4O C5sec
Gene SEQ ID NO: 5(HeavyMethyff assay)
rl~x:
Gagaggtttattaaggtaggg ( SEQ ID NO : 14584)
taaaaaattattaac (SEQ ID NO: 14585)
blocker:
tt8ttaaCStCC33caC8LttaLccaaacttce8a (SEQ ID NO:
14586)
probes:
8Wag8$Sdtgtagagaagttcga-fluo (SEQ ID NO: 14587)
-tcggataaacgcgtcgg-pho (SEQ ID NO: 14588)
LightCycler program:
'vati n: 95 C lOmin
197
CA 02528853 2005-12-23
c &10: 95 C 110 wc (20 C/s)
F- 56 C 130 sec (20 Cls) detection
72 C 110 sec (20 C/s)
f_95 C jlOsec 120
40 C 10soc 20
70 C losee 0,1
coolin~ 44 C 5 seo
Gene 6480 (HeavyMethyff assay)
Primer:
gagttgagagaatttttaaatttt ( SEQ ID NO : 14589)
Tcttctaaaacaaaaaacacc (SEQ ID NOt 14590)
Blocker:
Caccactacaatccaaacaacccaaa-pho (SEQ ID
NO: 14591)
Probes:
Ggacgtcgagcgttgggtat-fluo (SEQ ID NO:
14592)
Red640-aggacgcgtagttggacggt-pho (SEQ ID
NO: 14593)
' tCycler program:
95 C 10min
55 es: 95 C 10 sec 20 C/s)
56 C 30 sec 20 C/s) detection
72 C 10 soc (20 C/s)
1' c e: 95 C 10sec 20
40 C lOsec 20
70 C Osec 0,1
40 C 5 sec
198
CA 02528853 2005-12-23
Gme SEQ ID NO: 28 (HeavyMethyff assay)
Primer :
Gaaggaagggaaagtttt (SEQ ID NO: 14594)
Taaaaaaaacactacxctac (SEQ ID NO: 14595)
locker:
ctaccacaataaaaacccaaaacactcettcacacca-p ( S EQ ID NO :
14596)
Probe
acccgaaacgctocttcacg-fluo (SEQ ID NO: 14597)
red640-cgtaaacaaacgaaacgacgactacg Pho (SEQ ID NO :
14598)
LC program Roche :
de,gg-4t 95 C
95 10min
55 +cyples:
enat at 95 C 10 sec (20 CJs)
annealing 56 C 30 sec (20 C/s) etection
extension 72 C 10 sec (20 Cls)
e'
95 C lOsec 120
35 C 20sec 20
95 C 1(sec 0,1
Gene SEQ ID NO: 1(HeavyMethylm assaq)
otigos:
er:
Cacaaactatctataaaaaattaatctc ( SEQ ID NO : 14599)
ttgltgttggtgtttgttatt (SEQ ID NO : 14600)
locker:
ctcaaacacaaaaaaattaacatctcatetcttaaaccactttcaat-ph.o (SEQ
!ID NO: 14601)
Pmbes:
CgTCTCITA.AACCgCTTTCgA-fluo (SEQ ID N0:
j14602)
640-CCTCA.AAATAAACACgAA,CATACCC pho
199
CA 02528853 2005-12-23
(SEQ ID NO: 14603)
~
tLigLtCycler program:
vation: 95 C i0min
~ 1
1 95 C 110 sec 1(20 C/s)
56 C 130 sec (20 C/s) detection
72 C 10 sec (20 CIs)
curve: 95 C lOsec 120
40 C lOsec 0
70 C Osec 0,1
coalin~: 40 C 5 sec
Gene SEQ ID NO: 34 (HeavyMethyl7m assay)
Prixner:
Atacaaaa
onaataecttet (SEQ ID NO: 14604)
Atttggttttattttggttatg (SEQ ID NO : 14605)
~Blocker:
accttctcacaaatcaactaaaacacacacaacacc-pho (SEQ ID
NO: 14606)
Probes:
Red640-t88atttaS8Sc8tcScg-pho ( SEQ ID NQ:
14607)
ogttcgttmaagcggcga---IIuo (SEQ ID N4: 14608)
6tCycler program:
95 C 10min
95 C 10 sec (20 CIs)
56 C 130 sec (20 C./s) etection
72 C 10 sec (20 CJs)
e: 95 C lOsee 0
40 C 10sec 0
70 C Osec 10,1
00 40 C 5 sec
200
CA 02528853 2005-12-23
R-OSU+s
Sensitivity and specificity wera computed with SVM and 10 fold cross
validation
Table 7
Gene/Genomic
location AUC Sensitivity Specificity
Transcription 0.57 0.15 0.99
factor BTF3 Homolog (0.56)
1
RING FINGER PROTEIN 0.54 0.91 0.09
4 (0.55)
TUMOR NECROSIS 0.82 0.74 0.90
FACTOR RECEPTOR (0.82)
SUPERFAMILY MEMBER
16 PRECURSOR
Region of 0.83 0.69 0.94
chromosome 14q31.1 (0.84)
Chonclroitin sulfate 0.92 0.83 0.96
proteoglycan 2 (0.88)
(versican) (CPSG2)
0.83 0.71 0.94
EYA 4 (0.84)
MOTHERS AGAINST 0.54 0.25 0.91
DECAPENTAPLEGIC (0.52)
HOMOLOG 7 (SMAD 7)
ENDOPLASMIN 0.6 0.11 0.99
PRECURSOR (94 KDA (0.6)
GLUCOSE-REGULATED
PROTEIN) (GRP94)
(GP96 HOMOLOG)
(TUMOR REJECTION
ANTIGEN 1)
Region of 0.59 0.1 0.99
chromosome 6 25.1 (0.57)
B-CELI, LYMPHOMA 6 0.62 0.11 0.98
PROTFIN (BCL-6) (0.62)
(ZINC FINGER
PROTEIN 51) (LAZ-3
PROTEIN) (BCL-5)
HOMEOBOX PROTEIN 0.78 0.74 0.95
ARISTALESS-LIIKE 4 (0.75)
TRANSMEMBRANE 0.88 0.84 0.94
PROTEIN WITH EGF- (0.88)
LIKE AND TWO
FOLLISTATIN-LIKE
201
CA 02528853 2005-12-23
DOMAINS 2
Hypothetical 0.73 0.79 0.38
protein-leucine (0.72)
rich re eat
Candidate tumor 0.79 0.64 0.76
suppressor 8p22 (0.84)
VESICULAR 0.77 0.78 0.44
INHIBITORY AMINO (0.77)
ACID TRANSPORTER
(GABA AND GLYCINE
TRANSPORTER)
(VESICULAR GABA
TRANSPORTER)
(HVIAAT)
Sensitivity and specificity of multiple gene (panel) assays.
Panel: Assay 9 , Assay 12
Sensitivity: 0.88421052631579
Specificity: 0.972972972972973
Panelo Assay 11 , Assay 12
Sensitivity: 0.88421052631579
Specificity: 0.97027027027027
Panel: Assay 12 , Assay 15
Sensitivity: 0.9
Specificity: 0.95
Panel: Assay 6 , Assay 7
Sensitivity: 0.87
8pecificity: 0.973333333333333
Panel: Assay 5 , Assay 11
Sensitivity: 0.894736842105263
Specificity: 0.941333333333333
Panel: Assay 5, Assay 7
Sensitivity: 0.894736842105263
Specificity: 0.938666666666667
Panel: Assay 4 , Assay 6
Sensitivity: 0.857894736842105
Specificity: 0.971428571428571
Panel: Assay 7 , Assay 12
Sensitivity: 0.9052638EQ ID NO: 94737
Specificity: 0.921621621621622
Panel : Assay 6, Assay 12
202
CA 02528853 2005-12-23
Sensitivity: 0.842105263SEQ ID NO: 95
SpeCificity: 0.978378378378378
Panel: Assay 5 , Assay 9
Sensitivity: 0.842105263S$Q ID NO: 95
Specificity; 0.973333333333333
Panel: Assay 3, Assay 15
Sensitivity: 0.857142857142857
Specificity: 0.953424657534247
Panel: Assay 1 , Assay 12
Sensitivity: 0.842105263SBQ ID NO: 95
Specificity: 0.967567567567568
Panel: Assay 5 , Assay 15
Sensitivity: 0.857142857142857
Specificity: 0.945205479452055
Panel: SEQ ID NO: 17.2 , Assay 5
Sensitivity: 0.81764705882353
Specificity: 0.966233766233766
Panel: Assay 3 , Assay 12
Sensitivity: 0.8
Specificity: 0.983783783783784
Panel: Assay 6 , Assay 11
Sensitivity: 0.83SEQ ID NO: 947368421
Specificity: 0.952
Panel: Assay 5 , Assay 12
Sensitivity: 0.8105263SEQ ID NO: 9474
Specificity: 0.972972972972973
Panel: Assay 3, Assay 11
Sensitivity: 0.83SBQ ID NO: 947368421
Specificity: 0.949333333333333
Panel: Assay 3 , Assay 5
Sensitivity: 0.823529411764706
Specificity: 0.955844155844156
Panel: Assay 3 , SBQ ID NO: 17.2
Sensitivity: 0.8
Specificity: 0.974025974025974
Glass predictian bysupervised learning
In order to give a reliable estimate of how well the CpG ensemble of a
selected marker
set can differentiate between difl'erent tissue classes we determined its
prediction accuracy by
classification. For that purpose we calculated a methylation profile based
prediction function.
203
CA 02528853 2005-12-23
This step is called training and it exploits the prior knowledge represented
by the data labels.
The prediction accuracy of that funetion is then tested by cross-validation or
on a set of
independent samples. As a method of choice, we used the support vector machine
(SVM)
algorithm (Duda (2001), Christiannini (2000)) to learn the predictdon
funetion. Ifnot stated
otherwise, for this report the risk associated with false positive or false
negative classifications
are set to be equal relative to the respective class sizes. It follows that
the learning algorithm
obtains a class prediction function with the objective to optin-Aze accuracy
on an independent
test sample set. Therefore sensitivity and specificity of the resulting
classifier can be expected
to be approximately equal.Figures 87 to 95 show the performance of quadratic
SVMs trained on
the fall data set using the following assays.
Figure 87 :Assays 12 and 9
Figure 88 : Assays 12 and 11
Figure 89 : Assays 12 and 15
Figure 90: Assays 7 and 6
Figure 91 : Assays 1 I and 5
Figure 92: Assays 7 and 5
Figure 93 : Assays 6 and 4
Figure 94: Assays 12 and 7
Figure 95 : Assays 12 and 6
Ezamnle 15
HeavyMethyl anal is.
In the following, methylation of the genes/genonaic sequences according to
Gene SEQ ID
Nos: 7, 8 & 17 was analysed in 15 colorectal cancer samples (refen-ed to as
'colon' in Table 7),
and eight peripheral blood lymphocyte samples referred to as 'PBL' in Table
7).
Total genomic DNA of all samples was bisulfite treated converting unmethylated
cytosines to uracil. Methylated cytosines remained conserved. Bisulfite
treatment was performed
with minor modifications according to the protocol described in Olek et al.
(1996).
The sequence of interest was then amplified by means of primers specific for
bisulfite
treated DNA and a blocking oligonucleotide (in order to suppress amplification
of non-
methyla#ed DNA). The 3'OH terminus of the blocking oligonucleotide was
"capped" by a
phosphate group to prevent polymerase from extending the primer durintg PCR
reaction. The
amplificate i s t hen d etected b y m eans o f in ethylation s pecific
LightCyclerTm p robes, w herein
one probe was labelled with 5'red640 and had a capped 3'OH terminus and the
other was
204
CA 02528853 2005-12-23
labelled with 3'Fluorescein.
Genomic SEQ ID N0:8
Primers: aaaaaaaaaaaactcctctacatac (SEQ ID NO:14609)&
ggttattgtttgggttaataaatg (SEQ ID NO: 14610)
Blocking oligonucleotide: acatacaccacaaataaattaccaaaaacatcaaccaa
(SEQ ID NO: 14611+14612)
Probes : ttttttttttcggacgtcgtt (SEQ ID NO: 14613)
(Fluorescein) & tcggtcgatgttttcggtaa (red640) (SEQID NO: 14614)
Genomic SEQ ID NO:7
Primers : ggtttgggatggttgt(SEQ IDNO: 14615) &
tctaaataacaaaatacctccatt (SEQ ID NO: 14616)
Blocking ol igonucleot ides: ccattaccaacacaacccaccaaccaa (SEQ IDNO:
14617)
Probes : ggtcgcggtgagagagagag (Fluorescein) &(SEQ ID NO: 14618)
tgaaattagagcgcgtttcgg (red640) (SEQ IDNO: 14619)
Genomic SEQ ID NO:17
Primers : ggtgttgtttattttagagagtt (SEQ ID NO: 14620)
& cctaacccctatccttc (SEQ ID NO: 14621)
Blocking oligonucleotides: ccttcaccaccttcccaacactacaacac (SEQ II) NO: 14622)
Probes: tggggtttcggcgaagtaa (Fluorescein) (SEQ ID NO:14623) &
tttttcggcgtcggtcgt (red640) (SEQIDNO:14624)
The reactions were performed in a total volume of 20 1 using a LightCycler
device
(Roche Diagnostics). The real time PCR reaction mix contained 10 l of
template DNA, 2 l of
FastStart LightCycler reaction mix for hybridization probes (Roche
Diagnostics, Penzberg),
T 7: R sults of Hea eth 1 ass
al.cuiated
onceatratio
DNA
ndiluted
isaue roaai iate roaeing inta (/topl)
eaomiC SEQ ID iC S8Q ID or-iC 3 ID NO: 4 Gemomic SEQ ID NO: 4
205
CA 02528853 2005-12-23
O: 3 0: 12 (undiluted) (diluted)
oloa 36,73 38,22 39,58 5,855
olae 33,1 51 386,9
olon 39,8 42,84 57,5
olon 39,74 38,75 32,54 33,8 482,6
oloa 51 132,55
oloa 38,8 29,95S
oloa 32,08 33,9 33,43 35,43 816
oloa 37,03 36,9 284,35
ol= 38,07 46,54 0
oloa 35,22 37,81 36, 37,03 298,35
olon 33,66 36,19 33,23 34,0 1355
oloa 36,78 37,46 40,71 879
olon 37,23 57 5
oloa 1669
loa 34,88 34,71 34,21 34,4 880,5
PSL 716,5
aL 796
PSL 584,5
HL 38,55 1813
PSL 5470
3L 49,04 761
sL 625
F8L 39,7 9835
36,34 37,2 34,95 34,51. 583,5
100 36,06 37,86 34,64 34,09 428,3
F oche 1o0 34,48
hi 100
ter
Bold type figures indicate those classified as positive because the
amplification curves were flat.
Ezamale 15
5 Serum analyais of the genes acxording to SEQ ID Nos: 7. 30. 28 &8.
The assays as descrn.'bed above in example 13 (MSP) & 14 (HeavyMethyl) were
then applied
to a set of senun samples consisting 32 colorectal carcinoma serum samples,
and compared to a
set consisting of 54 normal senun samples, 11 serum samples from benign colon
lesions and 6
10 serum samples from intIanunatory colon disease. 130 ul of senmi was used
per assay and the
results were quantitatively reported as positive or negative.
Table 6
Marker or Sensitivity Specificity
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~9 :Ox CII S
rJMJVVVrJMVIVV,LJ,LJjrT,LJ
(9f9 :ox (n bas) (si :ox (n bas)
86f, W,L70.L,Lvw3'6'Ojl3O,L3w,La ,L
S~9 :Ox CQ S
00~'.L,LOa~~.L,LOO~,LO~,LLO
(1~~9 :Ox CllaaS) (~ :Ox CQ aEts)
0510 ZdVVJZJWV'~JD,LL1JVVJd',L,L
~~9 :Ox ca aS
o9VDVosDDdJL9d~v.LLoioLU
(Z~9 :ox cllaas) (uox QI bas)
vsz JaVVaDWDZVVWOaLO,L,LO
t~9 :oxQI as
owa aroodowaoo.i.Lvvv
(0~9 :ox cii Ws) (~i :ox (II Z3as)
6cz dJ0<JvV.LO<JVO<J,Ld,i,Vd,l,L,IJ.,L
6Z9 :ox Ca Oas)
,L,LLLLV,L,LSfJJ I.b'oWJJ1.,L2,L
(8Z9 :Ox CQ aas) (~uOx cllZ}aS)
9LV D3'd0vOyvo'Jo1.I.Yvd3vv
Z9 :ox Qa Oas)
00vK3VJ.O,L7,LdJõi.l'.LKaI3,LdO.I,
(9Z9 :Ox QI aas) (t i:Ox CII bas)
6b~ jJVJJVVD;J.L3õLLKDJW,L3õLV,L
SZ9 :ox (Q as
,LLJJZJIrJ,Ldr .t.LL,LvZJ LVrJ,L
(1-Z9 :ox cii bas) (t t :Ox (II aaS)
ZSti Z~d,I.Oa,LOt~'~ZO,L~.L~OOO,Ld
~Z9 :oxQI aS
T_TnT LT_TyyDVJ~-ZV',~J'H~tJJ,L
~ (zz9 :ox cclaas) (oi :ON ai bas)
:y uaZ
spollldwV =~~+d -aupJ
~Z-ZT-SOOZ ~588ZSZ0 Fi'J
ZiZ
OWW,Ld3o,I,D0OHDZ).LW.L3
(0L9 Ox (H aas) (OS :Ox cil Z?as)
9Z1r WVrJWdrJ,I.Ld,LJJ,L,i,W1J'~JYiJ
699 Ox CII a8
d.L,L'd.T.I.L,LvO.L,L,LaLO5aO,LL7
(899 :Ox (H aas) (oZ :ox CII aas)
s" ,Y.VDMVaGJV,LWZd',LOOD.LrJD
99 :ox CII as
0D01131=3VdJZV,LWd3VZ
(999 ox (31 bas) (LV :Ort cu bas)
t+86 ,L,LT.K.LJJJJJV,L,I.JD,LWJd.L'b'
s99 :ox cQ s
DO,L.L'b'VVW,L333LLdaWWV
(m:ox ai aas) (ZI :ox (Haas)
zob Jd,LVtJVxJDWVDJ.LJYJd,LJD1.,L
Q99 Ox aa as
D'~Jd'IV,Ld''~JV.LrJDDZ,LflE3WtJD
(Z99 :ox cil bas) (6 :Ox (II aas)
981 VVVV,La V,IO=,I,DV'b'WDdOO.L
i99 Ox ai as
J.IUJJV,I L,I,Vb'~'J;J1.,L,L,I,J,L,III.
(099 -Ox CII bas) (~1,:Ox cil aas)
OaL3w,L73J,O,T..733,LwZ3,L'4'
6S9 :ox al as
JJVDJJ'dD'~JbJVZJV,I.1.VW'OV
(859 :ox cil aas) (iz :ox m aas)
86~ VvW,LoO~DVKdX)W,La.T.o,LLs
S9 :Ox QI aS
DOOO1.,LW.LNIDDD1LaVZWW
(9s9 :ox aQ aas) (iz :ox (E bas)
oL~ ,LL,LJV1JWtJJbJrJ.LLLL,LtJW.L
SS9 :ON ca aS
,LDwJlJd,Lb'rJ1,floDZ,L.LtJ.L.Ld
(1-s9 :old cli bas) (~Z =ox (H aas)
68b rdd..LZLddõLaZb'J3"J30310
~s9 :ox QI as
IJV.L.I,VJ,LO<JW14.UdJJJJd
(zs9 :ox (H bas) (Lt :ox (H bas)
zsb 313131bOVOWDOWZDD.LW
TS9 :OAi QI aS
'~JV,L,~LJD,LLD.LD,LVJ'V()VJD,LL
(os9 :ox cii bas) (Lt :ox (H aas)
Z6V ,LIV'tTZV,L7ZOJOO,LOJ,LDddw
6ti9 :Ox al aS
VJ LJZWtl0000VJDZWxJ
(8b9 :Ox cg bas) (6i :ox cii bas)
sstb d'j3DSõLOb.L.La,LwwDDw'b'od
v9 =Oxaa as
.LO0O~~'o
ad'O~'~'tl~.LO'd
:q uaT
~lldurd :iaue~r~ :auag
~Z-ZT-SOOZ ~588ZSZ0 Fi'J
CA 02528853 2005-12-23
Gc,u: Prineer: Amplif:cate
Length:
(SEQ ID NO: 671)
CTCCAATTCTCCTCCCTATATC 425
(SEQ ID NO: 50) (SEQ ID NO: 672)
TAATTTTTGAGGTTGGGAAA
(SEQ ID NO: 673)
TABLE
Gene Olt o:
1 TGGACGTAGGAAAGCGA
(SEQ ID NO: 41) (SEQ ID NO: 681)
2 GATGTAGGAAAGTGAGA
(SEQ ID NO: 41 SE ID NO: 682
3 ATTTACGGGAGTTTTATCGT
(SEQ ID NO: 41) (UQ II) NO: 683)
4 ATTTATGGGAGTTITATTGT
(SEQ ID NO: 41) (SEQ II) NO: 684)
ATTAG'i TCGGGTCGCGT
(SEQ ID NO: 41) (SEQ ID NO: 685)
6 ATTAGTTTGGGTTGTGT
$ IDNO:41 (SEQ IDNO:
7 TATACGAAAGGGAGGCGG
SE ID NO: 41) (SEQ ID NO: 687)
8 TATATOAAAGGGAGGTGG
(SEQ ID NO: 41) (SDQ ID NO: 688)
9 GGCGTGTCGTTAGTTTTA
SE ID NO: 41) ID NO: 689)
GGTGTGTTGTTAGTTTTATA
SE ID NO: 41) (SEQ ID NO: 690)
11 TTCGATTGACGTTAGCGA
(SEQ ID NO: 41 (SEQ ID NO: 691)
12 TTTGATTGATGTTAGTGA
SE ID NO: 41) S ID NO: 692)
13 TTTCGAGTTTGACGGT
(SEQ ID NO: 41) (SEQ ID NO: 693)
14 TTTTGAGTTTGATGGTT
SE ID NO: 41) (SEQ ID NO: 694)
TTCGGAGGGCGTATTT
(SAQ ID NO: 41) SE ID NO: 695)
16 TTTGGAGGGTGTATTT
(SEQ II) NO: 41) (SEQ ID NO: 696)
17 GACGTCGGTACGTAGT
SE ID NO: 5) (SDQ ID NO: 697)
18 GATGTTGGTATGTAGTAG
SE ID NO: 5) SE IDNO: 698
19 TTCGGGGGAATTCGAGT
(SEQ ID NO: (SEQ ID NO: 699)
213
CA 02528853 2005-12-23
No: Gene Oti o:
20 TTTGGGGGAATTTGAGT
SE ED NO: 5) (SEQ ID NO: 700
21 TATTGCGAGGATTCGG
5E ID NO: 5) SE ID NO: 701)
22 ATTGTGAGGATTTGGT
(SEQ ID NO: 5) (SEQ ID NO: 702)
23 GTGCGTTCGTAGCGTA
(SEQ ID NO:
(Mg ID NO: 703)
24 TGTGTTTGTAGTGTAGG
SE ID NO: SE ID NO: 704
25 GGACGTCGTTTGTTAG
SE ID NO: (SEQ ID NO: 705)
26 GGATGTTGTT'TGTTAGG
SE ID NO: 5) (SEQ ID NO: 706)
27 AGAGCGTCGTTTTGTA
(SEQ ID NO: 5) (SEQ IL) NO; 707)
28 AGAGTGTTGTTTTGTAT
SE 1D NO: S ID NO: 708
29 TTTCGAGGGTAGGCGAG
SE ID NO: SE ID NO: 709
30 TTTTGAGGGTAGGTGAG
SE ID NO: 5) (SgQ ID NO: 710)
31 TTTCGAT'ITTAATGCGAA
SE ID NO: 5) (SEQ ID NO: 711)
32 TTTGATTTfAATGTGAAGT
SE II') NO: 5) SE ID NO: 712)
33 AGGAATTTCGTCGCGA
SE ID NO: SB ID NO: 713
34 AGGAATTTTGTTGTGAT
(SEQ ID NO: 5) (SEQ ID NO: 714
35 T'TTGAGTCGTACGCGT
(SEQ ID NO: SE ID NO: 715
36 TTTTGAGTTGTATGTGT
(SEQ ID NO: (SAQ ID NO: 716
37 TACGTAGTTGCGCGTT
SE ID NO: SE IDN0:71
38 GTATGTAGTTGTOTGTT
(SEQ ID NO: 51 SB ID NO: 674)
39 AATCGGCGGTTAGGAT
SE ID NO: (SEQ ID NO: 718
40 GAATTGGTGGTTAGGA
SE ID NO: SE ID NO: 719
41 TTTGATCGGGTTTGAG
SE ID NO: CSEQ ID NO: 720
42 TTT I'GATTGGGTTTGAG
SE ID NO: SE ID NO: 72b
43 TTTGAGTATTCGTAGGAA
SE ID NO: 51 (SEQ ID NO: 675)
44 TGAGTATTi'GTAGGAAGA
(SEQ ID NO: SE ID NO: 722
214
CA 02528853 2005-12-23
No: Gexe Ol =
45 AGAGGCGCGGGTTATA
(SAQ ID NO: 6) SE ID NO: 723)
46 TAGAGGTGTGGGTTAT
ID NO: SE ID NO: 724
47 TTAGCGGTTAAGTTGCGA
SE ID NO: 6) (SEQ ID NO:
48 TTAGTGGTTAAGTTGTGA
SE ID NO: SB ID NO: 72
49 TTCGTAGAAGAATACGCGTA
(SEQ ID NO: 8) (SEQ ID NO: 727)
50 TTTGTAGAAGAATATGTGTA
(SEQ ID NO: 8) (SEQ ID NO: 728)
51 AAACGTTTATCGGTTG
SE ID NO: 8 (SEQ ID NO: 729)
52 AATGTTTATTGGTTGGA
SB ID NO: 8) (SEQ ID NO: 730)
53 TATCGTAGTTCGTTCGG
(SEQ ID NO: 8) (SEQ II) NO: 731)
54 ATTGTAGTTTGTTTGGT
(SEQ ID NO:8 S ID NO: 732
55 TGGTCGGTATATTTCGA
SE ID NO: 16) SE ID NO: 733)
56 TTGGT'1'GGTATATTTTGA
SE ID NO: 1(SEQ ID NO: 734)
57 GGAGGTTTCGGTTCGA
(SEQ ID NO: 16) (SEQ ID NO: 73
58 TGGAGGT'1"I"'TGGTTTGA
SE ID NO:1 SE ID NO: 73
59 TTAGCGGTAATAGCGG
(SEQ ID NO: 16) SE ID NO: 737)
60 TATTAGTGGTAATAGTGG
(SEQ ID NO: 52) SE ID NO: 676)
61 TGCGTAGTAGGCGGTT
SE ID NO: 42) (SEQ II) NO: 738)
62 TGTGTAGTAGGTGGTTT
SE IDNO: 53) (SEQ ID NO: 677)
63 TAGGCGGTTGTTCGTA
F S ID NO: 42) (SEQ ID NO: 739)
64 AGGTGGTT'GTTTGTAA
SE ID NO: 42 (SEQ ID NO: 740)
65 TTGAAGTCGGTACGGT
SE ID NO: 14) (SEQ ID NO: 1125)
66 TGAAGTTGGTATGGTT
SE ID NO: 14) SE ID NO: 1 i26
67 TGGGACGCGGATATTT
SE ID NO: 14) SE ID NO: 1127)
68 GTTGGGATGTGGATAT
SE ID NO: 14) (SEQ ID NO: 1128
69 GTTCGGGTCGATTCGA
SE ID NO: 43)
SE ID NO: 741 '
215
CA 02528853 2005-12-23
No: Gene Oli o:
70 GGTT'XGGGTTGATTTGA
ID NO: 43) SE ID NO: 742)
71 TTCGGGATATATTCGATT
(SEQ ID NO: 43) SE ID NO: 743)
72 TTTTGGGATATATTTGATT
(SEQ ID NO: 43) SE ID NO: 744
73 TATTCGAATTGTATTCGT
(SEQ ID NO: 43) (SEQ ID NO: 745)
74 TTTGAATTGTATTTGTTTAT
ID NO: 43) SE ID NO: 746)
75 TTAAGTTCGATTCGGT
SE ID NO: 15(SEQ ID NO: 747)
76 AGTTTGATTTGGTGAAT
S ID NO: 54) (SEQ ID NO: 678)
77 TAGTTGTTCGAGAGGG
SE ID NO: 15) (SEQ ID NO: 748)
78 AGTTGTT'TGAGAGGGT
(SEQ ID NO: 15) (SEQ ID NO: 749
79 ATAGTATCGAGGTGAGT
(SEQ ID NO: 15) SE II? NO: 750)
80 ATAGTATTGAGGTGAGTT
SB ID NO: 15) SE ID NO: 751)
81 TTCGGGATTCGATAAT
SE ID NO: 15) (SEQ ID NO: 752)
82 AGGTTTTGGGAT'ITGA
(SEQ ID NO: 15) SE ID NO: 753
83 TTAGGACGCGGCGATA
(SEQ ID NO:1S (SEQ 1D NO: 754)
84 AGGATGTGGTaATAGT
ID NO: 15) (SEQ ID NO: 755)
85 TTGTACGTTCGGTATT
S ID NO: 15) (SEQ ID NO: 756)
86 TAAGTTGTATGTTTGGfA
SE ID NO: 15(SEQ ID NO: 757)
87 GGCGTCGAGGTCGTAG
(SEQ ID NO: 4 S ID NO: 758)
88 GGTGTTGAGGTTGTAGT
(SEQ ID NO: 4) (SEQ ID NO: 759)
89 AGGGTTTCGATTTTCGG
(SEQ ID NO: 4) S ID NO: 760
90 AGGGTTTTGATTTTTGG
SE ID NO: 4) (SEQ ID NO: 761
91 TGGAACGTGCGATTGT
(SEQ ID NO: 4) (SEQ ID NO: 762)
92 TGGAATGTGTGATTGT
(SEQ ID NO: 4) SE ID NO: 763)
93 TTT"PGGCGCGI'TTATA
( EQ ID NO: 4) S ID NO: 764)
94 TTGGTGTGTTTATAGATA
SE ID NO: 4 (SEQ ID NO: 765)
216
CA 02528853 2005-12-23
No: Gene Ol' :
95 AGATTTTTACGATTCGA
S ID NO: 4 S ID NO: 766
96 TTTTTATGATTTGAAATAGA
(SEQ ID NO: 4) (SEQ ID NO: 767)
97 AGTATTTTCGCGTGTT
SE ID NO: 4) (SEQ ID NO: 768)
98 TAGTAT"P'FTTGTGTGTTAA
SE ID NO: 4) SE ID NO: 769)
99 TTCGTCGGCGGTAGAG
(SEQ ID NO: (SEQ ID NO: 770)
100 TAGAGTTTGTTGGTGG
(SEQ ID NO: (SEQ ID NO: 771
101 GATCGCGGGTACGTTT
S ID NO: (SEQ ID NO: 772
102 ATTGTGGGTATGTTTGT
(SEQ ID NO: (SEQ ID NO: 773)
103 TTAACGTCGTTGGTTA
S ID NO: S ID NO: 774
104 TGATTAATGTTGTTGGT
S ID NO: (SEQ ID NO: 775)
105 TTCGCGCGAAGATTTA
(SEQ ID NO: (SEQ ID NO: 77
106 GTT'I"TTGTGTGAAGATT
S ID NO: (SEQ ID NO: 777)
107 TTCGATATCGTGACGG
ID NO: 44) SE ID NO: 778
108 TTTTGATATTGTGATGGT
S ID NO: 44) S ID NO: 779
109 AGAATACGGTCGTAGA
(SEQ ID NO: 44) (SEQ ID NO: 780
110 TAGAATATGGTTGTAGATA
SE ID NO: 44) S ID NO: 781)
111 TATTTTfGCGTACGGG
ID NO: 44) S ID NO: 782)
112 ATTTTTGTGTATGGGTT
SE ID NO: 44) (SEQ ID NO: 783)
113 - TTACGGTGAAGGCGGA
S ID NO= 1) S ID NO: 784
114 TTATGGTGAAGGTGGA
S ID NO: 1) (SEQ ID NO: 785
115 TTCGGGATrAATATCGAGAT
ID NO:1 S ID NO: 7
116 TTTGGGATTAATATTGAGAT
(SEQ ID NO: 1(SEQ ID NO: 7
117 TTTCGGTTTTCGTTAAT
(SEQ ID NO: 1(SEQ ID NO: 788)
118 TTTGGTTTTTGTTAATTTAG
(SEQ ID NO: 1SE ID NO: 789
119 TGTGCGAAGTTAACGT
(SEQ ID NO:1 S IDNO: 790
217
CA 02528853 2005-12-23
No: Gene O o:
120 TTGTGTGAAGTTAATGT
SE ID NO:1 (SEQ ID NO: 791)
121 AAGTTTATCGGCGTTT
(SEQ ID NO: 1) SE ID NO: 792)
122 AGAAGTTTATTGGTGTTT
SE ID NO: 1ID NO: 793)
123 ATTTCGGAATTTAAGCGT
SE ID NO: 1) (SEQ ID NO: 794)
124 TTTGGAATTTAAGTGTTT
(SEQ ID NO: 1(SEQ II) NO: 795)
125 TTTTCGCGATTGGAGA
(SEQ ID NO:1 S ID NO: 7
126 GTT ITTGTGATTGGAGA
(SEQ ID NO: 1) (SE_Q ID NO: 797)
127 ATTTACGCGTTTTAGG
S ID NO: 1(SEQ ID NO: 798)
128 ATGGAATTTATGTGTTT"T
(SEQ ID NO: 1) SE IDNO: 799
129 ATGTCGCGGTTTTATA
ID NO: 1(SAQ ID NO: 800
130 GGATGTTGTGGTTTTAT
(SEQ ID NO: 1) SE ID NO: 801)
131 AGACGGGGTTTACGAG
(SEQ ID NO: 2) (SEQ ID NO: 802)
132 AGATGGGGTTTATGAG
S ID NO: 2) S ID NO: 803)
133 TGTCGGTATTAGCGTT
(SEQ ID NO: 2) S ID NO: 804)
134 GTGTTGGTATTAGTGTT
SE ID NO: 2) S ID NO: 805)
135 TGGTTTACGTTCGGTA
(SEQ TD NO: 2) $ID NO: 806)
136 GGTTTATGTTTGGTAGT
SE ID NO: 2) SE ID NO: 80
137 TTCGTACGGTTAGGTT
(SEQ ID NO: 2) (SEQ ID NO: 808)
138 AGTTTTGTATGGTTAGG
S ID NO: 2 (SEQ ID NO: 809)
139 ATAGCGATTTCGGCGA
(SEQ ID NO: 45) S ID NO: 810
140 AGTGATTTTGGTGAGA
SE ID NO: 45) S ID NO: 811)
141 GGCCTTT"TATTTACGAGA
(SEQ ID NO: 45) (SEQ ID NO: 812)
142 GGGTGTTTTATTTATGAG
SE ID NO: 45) ID NO: 813
143 ATCGTGGACGGTA.ACGA
(SEQ ID NO: 45) (SEQ ID NO: 814)
144 ATTGTGGATGGTAATGA
CSEQ ID NO: 45 (SEQ ID NO: 815)
218
CA 02528853 2005-12-23
No: Gene Olt o:
145 TTGAGATCGATTCGTT
SE ID NO: 45 S Il) NO: 816)
146 TGAGATTGATTTGTTTAG
(SEQ ID NO: 45) SE IDNO: 81
147 GGCGAGATTCGTACGT
SE ID NO: 45) (SEQ ID NO: 818)
148 GGTGAGATTTGTATGI?'
SE ID NO: 45) (SEQ ID NO: 819)
149 TGACGTTCGTGGTGGA
(SEQ ID NO: 45 (SEQ ID NO: 820
150 GATGTTTGTGGTGGAG
SE ID NO: 45) SE ID NO: 821)
151 GTGATCGATTACGGTA
(SEQ ID NO: 45) ID NO: 822
152 AGGTGATTGATTATGGT
SE ID NO: 45) ID NO: 823)
153 ATTATTCGTTCGGTGA
(SEQ ID NO: 45) S ID NO: 824)
154 TATTATTTGTTTGGTGAG
S ID NO: 45) (SEQ ID NO: 82
155 TATCGTCGTTAAGTGT
(SEQ ID NO: 45) ll.) NO: 826)
156 TATTATTGTTGTTAAGTGT
(SEQ ID NO: 45) ID NO: 827)
157 TGTAAGCGCGAGAATA
(SEQ ID NO: 45) II) NO: 828
158 AGTGTAAGTGTGAGAAT
SE ID NO: 45) ID NO: 829
159 TATAGCGGTTTACGGT
(SEQ ID NO: 9) (SEQ ID NO: 830
160 TAGTGGTTTATGGTAGT
(SEQ ID NO: 9) SE ID NO: 831
161 AG(',GCGATTAGGACGT
SE ID NO: 9) (SEQ II? NO: 832)
162 AC',GGTGATTAGGATGT
(SEQ ID NO: 9 (SEQ ID NO: 833)
163 TTCGTTAGAGTTCGTAG
SE ID NO: ID NO: 834)
164 TTTGTTAGAGTTTGTAGT
(SEQ ID NO: (SEQ ID NO: 835)
165 TGAGACGTTTGTCGGT
SE ID NO: S ID NO: 836
166 TGAGATGTTTGTTGGT
SE ID NO: (SEQ ID NO: 837)
167 GAAAAGTTCGTCGGTT
SE ID NO: (SEQ ID NO: 838
168 AGAAAAGTTTGTTGGTT
SE ID NO: (SEQ ID NO: 839)
169 ATGGCGTAGTCGCGAT
S ID- NO: SE ID NO: 840
219
CA 02528853 2005-12-23
No: Gene Ol' o:
170 TGGTGTAGTTGTGATT
S ID NO: 46) (SEQ ID NO: 841)
171 TTTTGACGTCGATGTA
S ID NO: 10) SE ID NO: 842)
172 TGATGTTGATGTAGAATT
(SEQ ID NO: 10) SE JOD NO: 843)
173 TTGCGATGTGCGTTTA
(SEQ ID NO: 10) (SEQ I[) NO: 844
174 TGTGATGTGTGTTTAGT
S ID NO: 10) (SEQ ID NO: 845)
175 TGATTACGGCGCGGAT
SE ID NO: 10) S ID NO:
176 ATTATGGTGTGGATGG
(SEQ ID NO: 10 (SEQ ID NO: 847)
177 AGATGGCGACGTCGAA
SE ID NO: 10 (SEQ ID NO: 848)
178 ATGGTGATGTTGAAGA
SE ID NO: 10 (SEQ ID NO: 849
179 TTTAAGCGCGGCGGTA
(SEQ ID NO: 10) (SEQ ID NO: 850)
180 TTTTAAGTGTGGTGGTA
SE ID NO: 10) (SEQ ID NO: 851
181 AGAAACGTAGACGCGA
(SEQ ID NO: 10 (SEQ ID NO: 852)
182 AATGTAGATGTGATGGA
(SEQ ID NO: 10 (SEQ ID NO: 853)
183 AGAGACGCGAAAAATT
(SEQ ID NO: 11) (SEQ ID NO: 854)
184 TAGAGAGATGTGAAAAAT
(SEQ ID NO: 11) (SEQ ID NO: 855)
185 AGACGAAAGAGTCGTT
(SEQ ID NO: 11) (SEQ ID NO: 856)
186 AGAGATGAAAGAGTTGT
(SEQ ID NO: 11) S ID NO: 857)
187 TTTTAGTTCGAGCGTA
(SEQ ID NO: 11) SE ID NO: 858)
188 TTAGTTTGAGTGTAGTTA
(SEQ ID NO: 11) (SEQ ID NO: 859
189 GACGTGAATT'I"TCGGAA
(SEQ ID NO: 11) S ID NO: 860)
190 AGGATGTGAATTTTTGG
(SEQ ID NO: 11) (SEQ ID NO: 861)
191 AATGCGTGGTCGTTT"T
(SEQ ID NO: 11) SE ID NO: 862)
192 GAAATGTGTGGTTGTT
(SEQ ID NO: 11) (SEQ ID NO: 863
193 TTTCGTTTGCGGAATT
(MQ ID NO: 11) (SEQ ID NO: 864)
194 G
(SEQ SE ID NO: 11) (SE ID NO: 865)
220
CA 02528853 2005-12-23
=
No: Gene Oli o:
195 TGTTCGACGTGATTTT
SE ID NO: 12 SE ID NO: 960
196 GTGTTTGATGTGATTTT
(SEQ ID NO: 12(SEQ ID NO: 867)
197 TAACGTTTTTTCGGGT
SE ID NO:12 (SEQ ID NO: 868)
198 TTAATGIITTTTTGGGTG
SE ID NO: 12) (SEQ ID NO: 869)
199 TGTTGATTCGGAAATGA
(SEQ ID NO: 12(SEQ ID NO: 870
200 TTTGTTGATTTGGAAATG
(SIBQ ID NO: 12SE ID NO: 871)
201 TAAAGTTTCGAAGCGG
SE ID NO: 13SE ID NO: 1129)
202 AG'TI"I"TGAAGTGGAGT
(SEQ ID NO: 13) (SEQ ID NO: 1130)
203 AAGTCGGTAGTTATCGT
CSIBQ ID NO:13 (SEQ ID NO: 1131)
204 AAGTTGGTAGTTATTGTT
(SEQ ID NO: 13(SEQ ID NO: 1132)
205 TTGGAGCGCGAGAAAG
SE ID NO: 3) (SEQ ID NO: 872)
206 TTGGAGTGTGAGAAAG
SE ID NO: 3) (SEQ ID NO: 873)
207 TACGTTATCGGTTCGT
(SEQ ID NO: 3) (SAQ ID NO: 874)
208 TATGTTATTGGf'I"fGTATT
(SEQ ID NO: 3 SE ID NO: 875)
209 ATTAGGTTCGTGGGCGT
(SAQ ID NO: 3) SE ID NO: 876)
210 ATTAGGTTTGTGGGTGT
(SEQ ID NO: 3) S ID NO: 87
211 TGCGGTTTAGAAACGTAG
(SEQ ID NO: 3) (SEQ ID NO: 878)
212 TGTGGTTTAGAAATGTAG
SE ID NO: 3) (SEQ ID NO: 879)
213 GAACGGGTTTCGTAGT
SE ID NO: 3) (SEQ ID NO: 880
214 GGGAATGGGTTTTGTA
S ID NO: 3) (SEQ ID NO: 881
215 TTGCGATAGTCGGCGG
(SEQ ID NO: 3) (SEQ ID NO: 882)
216 TTGTGATAGTTGGTGG
SE ID NO: 3) (SEQ ID NO: 883)
217 AAGAACGGACGTGTTT
(SEQ ID NO: 3) (SEQ ID NO: 884)
218 AGGAAGAATGGATGTG
SE ID NO: 3) (SEQ ID NO: 885)
219 AAGTTTCGTTTGGGAG
SE ID NO: 47) (SEQ ID NO: 886
221
CA 02528853 2005-12-23
No: Gexe Ol' o:
220 AAAGTTTTGTTTGGGAG
S ID NO: 47) (SEQ ID NO: 887)
221 TTGGAAGTCGAAGAGA
(SEQ ID NO: 47) (SEQ ID NO: 888)
222 TTTGGAAGTTGAAGAGA
(SEQ ID NO: 47) (SEQ ID NO: 889)
223 TATCGGGTTCGATTTT
(SEQ ID NO: 1 (SEQ ID NO: 890)
224 GGTGTATTGGGTTTGA
(SEQ ID NO: 18) (SEQ ID NO: 891)
225 TAGGGATTCGCGGAGG
(SEQ ID NO: 20) (SEQ ID NO: 892)
226 TAGGGATTTGTGGAGG
(SEQ ID NO: 20) (SEQ ID NO: 893)
227 TTGTCGAGTAATTTTCGT
(SEQ ID NO: 20) (SEQ ID NO: 894)
228 TGTTGAGTAATTTTTGTT
S ID NO: 20) (SEQ ID NO: 895)
229 TATTACGGGCGGAGGG
(SEQ ID NO: 20) (SEQ II) NO: 896)
230 TATTATGGGTGGAGGG
(SEQ ID NO: 20) (SEQ ID NO: 897)
231 GACGGTACGTTAGAGG
(SEQ ID NO: 20) (SEQ ID NO: 898)
232 GATGGTATGTTAGAGGT
(SEQ ID NO: 20) SE ID NO: 899
233 TTGGGCGTCGTTATTA
(SEQ ID NO: 20) (SEQ ID NO: 900)
234 TGGGTGTTGTTATTAGT
(SEQ ID NO: 20) (SEQ ID NO: 901)
235 TATTAGTTCGGTCGTT
(SEQ ID NO: 20) (5_EQ ID NO: 902)
236 AGTTTGGTTGTTAGTTT
(SEQ ID NO: 20) (SEQ ID NO: 903)
237 TTATTACGTTTAGCGAT
(SEQ ID NO: 20) (SEQ ID NO: 904)
238 TTTTTATTATGTTTAGTGATA
(SEQ ID NO: 20) (SEQ ID NO: 905)
239 ATAGCGAGTGCGATAT
(SEQ ID NO: 20 (SEQ ID NO: 906)
240 AGGGTCGTAGCGGTAG
(SEQ ID NO: 48) SE ID NO: 907)
241 GAGGGTTGTAGTGGTA
SE ID NO: 48) (SEQ ID NO: 908
242 TTAGGTCGGACGTAAG
SE ID NO: 50 (SEQ ID NO: 1143)
243 GGTTAGGTTGGATGTA
SE ID NO: 50) (SEQ ID NO: 1144)
244 TAGACGTGGGGTTACGT
(SEQ ID NO: 22) (SEQ ID NO: 909)
222
CA 02528853 2005-12-23
No: Gene O!' .
245 TAGATGTGGGGTTATGT
SE ID NO: 22) SE II? NO: 910
246 ATTTCGG'fiGTAGTATCGT
(SEQ ID NO: 22) (WQ ID NO: 911
247 ATTTTGGGGTAGTATTGT
(SEQ ID NO: 22) SE ID NO: 912)
248 TACGCGCGTTTTAAAA
SE ID NO: 19) (SEQ ID NO: 913)
249 TTATGTGTGTTTTAAAATG
SE ID NO: 19 S ID NO: 914
250 TACGATATCGTTATATAACGG
SE ID NO: 19 (SEQ ID NO: 915)
251 TATGATATTGTTATATAATGG
SE ID NO: 19) (SEQ ID NO: 91
252 TATAGGTTCGCGGTTT
(SEQ ID NO: 19) (SEQ ID NO: 917)
253 TATATAGGTTTGTGGTTT
SE ID NO:19 ID NO: 918)
254 TAGGTGCGCGTTATAT
(SEQ ID NO: 19) (SEQ ID NO: 919)
255 ATGTAGGTGTGTGTTAT
SE ID NO: 19SE ID NO: 920)
256 TACGTTGTTTGGACGAAT
(SEQ ID NO: 55) SE ID NO: 679)
257 TATGT'TGTTTGGATGAAT
(SEQ ID NO:19 SE ID NO: 921
258 AAGGAGCGTATTTCGG
($EQ ID NO: 19SE ID NO: 922)
259 AGGAGTGTATTTTGGG
(SEQ ID NO: 19) (SRQ ID NO: 923)
260 GTCGGATTTCGGAAGT
SE ID NO: 55) SE II) NO: 680)
261 GTTGGATTTTGGAAGTG
SE ID NO: 19) ID NO: 924)
262 GAAGTGACGCGTTCGT
SE ID NO:19 SE ID NO: 925)
263 GAAGTGATGTGTTTGT
(SEQ ID NO: 19 (SEQ ID NO: 926)
264 TGTTATCGTTGCGCGA
SE ID NO: 19) (SgQ ID NO: 927)
265 ATGTTATTGTTGTGTGA
(SEQ ID NO: 19SE ID NO: 928)
266 TGAAAACGTTTT"TCGT
SE ID NO: 17) SE ID NO: 929)
267 AATGTT'!"ITTGTAAAGAAA
SE ID NO: 1SE ID NO: 930
268 AGGATTTCGGCGTTAT
(SEQ ID NO: 17) SE
Q ID NO: 931
269
F AAAGGATTTTGGTGTTA
SE ID NO: 1 SE ID NO: 932)
223
CA 02528853 2005-12-23
No: Gene OI' o:
270 ATTTATTCGTGCGTTT
(SEQ ID NO: 17) (SEQ ID NO: 933)
271 TATTTGTGTGTTTAGGG
SE ID NO: 934)
(SEQ ID NO: 17
272 TTTCGGTGGTTTTCGAA
(SgQ ID NO: 17) (SEQ ID NO: 935)
273 TTTGGTGGTTTTTGAAG
(SEQ ID NO: 17) (SEQ ID NO: 936)
274 GGCGTACGGAATTTTA
(SEQ ID NO: 17) (SEQ ID NO: 937)
275 GGGTGTATGGAATTTTA
(SEQ ID NO: 17) (SEQ ID NO: 938)
276 TGGACGGAGGTTTCGT
(SEQ ID NO: 17) (SEQ ID NO: 939)
277 TGGATGGAGGTTTTGT
S ID NO: 17) SE ID NO: 940)
278 TGCGGACGGGATAGTT
(SEQ ID NO:1 (SEQ ID NO: 941)
279 TGTGGATGGGATAGTT
SE ID NO: 1 (SEQ ID NO: 942)
280 TGATTAGTCGATTCGT
(SEQ ID NO: 17) SE ID NO: 943)
281 GATGTAGGGATGGAGA
(SFQ ID NO: 17) (SEQ ID NO: 944)
282 TATCGTGGTTTTTTACGTAT
(SEQ ID NO: 17) (SEQ ID NO: 945)
283 ATATTGTGGTTTTTTATGTA
(SEQ ID NO: 17 (SEQ ID NO: 946)
284 TTTATTCGGTGTTCGA
SE ID NO: 17) (SEQ ID NO: 947)
285 TATTTGGTGTTTGAGAG
(SEQ ID NO: 17) (SEQ ID NO: 948)
286 GAGGCGCGTTAT"I"T'?'T
$E ID NO: 23) (SEQ ID NO:1133
287 GGGAGGTGTGTTATTTT
(SEQ ID NO: 23) (SEQ ID NO: 1134)
288 AACGGTAGTTAGCGATA
(SEQ ID NO: 23) (SEQ ID NO: 1135)
289 TGAATGGTAGTTAGTGA
(SEQ ID NO: 23) (SEQ ID NO: 113
290 TTTTAACGTTCGCGGA
SE ID NO: 23) (SEQ ID NO: 1137
291 AATGTTTGTGGAGGAT
SE ID NO: 23) (SEQ ID NO: 1138
292 TTTTTCGCGTATATGTT
(SEQ ID NO: 21 (SEQ ID NO:1139
293 TT'TTTTGTGTATATGTTTAGG
SE IDNO: 21 (SEQ ID NO: 1140
294 AAGGGCGGTAAGACGG
SE ID NO: 21) (SEQ ID NO: 1141)
224
CA 02528853 2005-12-23
No: Gene 4 0:
295 AAGGGTGGTAAGATGG
SE ID ND: 21) SE ID NO: 1142
296 GGTTTCGTTTAATCGT
(SEQ ID NO: 32) (SEQ ID NO: 949)
297 GGGTTTTGTTTAATTGTA
(SEQ ID NO: 32) (SEQ ID NO: 950
298 GATTCGTATTTCGTAGT
S ID NO: 32) SE ID NO: 951)
299 TTTGTATTTTGTAGTGGG
(~WQ ID NO: 32) (SIRQ ID NO: 952)
300 TTCGTATTTAGCGGAT
(SEQ ID NO: 32) (SEQ ID NO: 953)
301 GGTTTGTATTTAGTGGA
(SEQ ID NO: 32 S ID NO: 954
302 TTAATCGGCGGGTTTT
S ID NO: 32 (SEQ ID NO: 955)
303 AGTTAATTGGTGGGTT
(SEQ ID NO: 32 (SEQ ID NO: 95
304 TATTTTGGCGGGTTGTAT
(SEQ ID NO: 32) (SEQ ID NO: 957)
305 TATTTTGGTGGG'I'TGTAT
(SEQ ID NO: 32) SB ID NO: 958)
306 AAGGTTATCGGTTTAAGA
SE ID NO: 32) (SEQ ID NO: 959)
307 AAGGTTATTGt'irTTTAAGA
SE ID NO: 32 SE ID NO: 960
308 GGGGGACGACGTTTTTGT
SE ID NO: 32 SE ID NO: 961)
309 GGGGGA.TGATGT'TTT TGT
(SEQ ID NO: 32) SE ID NO: 962)
310 TTACGGTTCGGTTATT
(SEQ ID NO: 33) S ID NO: 963)
311 AGGTTTTATGGTTTGGT
(SEQ ID NO: 33) (SEQ ID NO: 964)
312 GACGTCGCGGGGTTAG
(SEQ ID NO: 33) (SEQ II) NO: 965)
313 TGATGTTGTGGGGTTA
(SEQ ID NO: 33) E ID NO: 966)
314 AGGTATTTCGCGATAT
(SEQ ID NO: 33) (SEQ ID NO: 967)
315 AGGTATTTTGTGATATTI't
(SEQ ID NO: 33 (SEQ ID NO: 968)
316 GTTI'TTCGATTTACGTT
(SEQ ID NO: 33) SE ID NO: 969
317 TAGGTTTTTTGATTTATGT
(SEQ ID NO: 33) SE ID NO: 97Q
318 GGTAGTTTCGATTATTTA
(SEQ II? NO: 33 (SgQ ID NO: 971)
319 GGTAGTTTTGATTATTTA
(SEQ ID NO: 33 SE Il? NO: 972
225
CA 02528853 2005-12-23
No: Gene Oli o:
320 TAGAGTACGGGGCGGG
(SEQ ID NO: 33) (SgQ ID NO: 973
321 TAGAGTATGGGGTGGG
(SEQ ID NO: 33) (SEQ ID NO: 974)
322 TTGTTTAGCGGATTTTAG
(SEQ ID NO: 33) SE ID NO: 975)
323 TTGTTTAGTGGATTTTAG
(SEQ ID NO: 33) (SEQ ID NO: 976)
324 TAGGTTCGGTTCGTTAT
(MQ ID NO: 33) (SEQ ID NO: 977)
325 TAGGTTTGGTTTGTTATT
(SEQ ID NO: 33) (SEQ ID NO: 978
326 TGGTGGTACGTAGTTTGG
(SEQ ID NO: 33) (SEQ ID NO: 979)
327 TTTGGCGTAGATCGGT
(SEQ ID NO: 33) (SEQ ID NO: 980
328 TTTGGTGTAGATTGGT
(SEQ ID NO: 33) (SEQ ID NO: 981)
329 AGTGTTCGTCGTAGTT
(SEQ ID NO: 33) (SEQ ID NO: 982)
330 TGAGTGTTTGTTGTAGT
SE ID NO: 33) (SEQ ID NO: 983)
331 GTGTTTAGCGCGGATT
(SEQ ID NO: 33) (SEQ ID NO: 984)
332 GGTGTTTAGTGTGGATT
(SEQ ID NO: 33) (SEQ ID NO: 985)
333 TTCGGTTAGTTTCGTAT
(SEQ ID NO: 34) (SEQ ID NO: 986)
334 TTTTGGTTAGTTTTGTATT
(SEQ ID NO: 34) (SEQ ID NO: 987)
335 GATTCGTTTGGGTAACGT
(SEQ ID NO: 34) S ID NO: 988
336 GATTTGTTTGGGTAATGT
(SEQ ID NO: 34) (SEQ ID NO: 989
337 GTCGAATTTAGTCGCGT
(SEQ ID NO: 34) (SEQ ID NO: 990
338 GTTGAATTTAGTTGTGTA
(SEQ ID NO: 34) (SEQ ID NO: 991)
339 AATTCGCGAGTTTAGA
(SEQ ID NO: 34) (SEQ ID NO: 992)
340 A.A.AAATTTGTGAGTTTAG
SE ID NO: 34) (SEQ II) NO: 993)
341 AGGGGTTCGATTAGGG
SE ID NO: 24) (SEQ ID NO: 1145)
342 AGGGGTTTGATTAGGG
(SEQ ID NO: 24) (SEQ ID NO: 11
343 TTAGGTATACGAAAGAGTAT
(SEQ ID NO: 24) S II) NO: 1147)
344 TTAGGTATATGAAAGAGTAT
(SEQ ID NO: 24) (SEQ ID NO:1148
226
CA 02528853 2005-12-23
No: Gene OI o:
345 TGTCGTACGTTATGTT
SE ID NO: 24) (SEQ ID NO: 1149)
346 GGTGTTGTATGTTATGT
(SEQ ID NO: 24) (SEQ ID NO: 1150)
347 TTGATTGGCGGACGAG
(SEQ ID NO: 24 (SEQ ID NO: 1151
348 TTGATTGGTGGATGAG
(SEQ ID NO: 24) (SEQ ID NO: 1152
349 TATATATACGTGTGGGTA
(SEQ ID NO: 35) (SEQ ID NO: 994)
350 TATATATATGTGTGGGTA
(SEQ IA NO: 35) (SEQ ID NO: 995)
351 TATGTAGTCGCGTAGT
(SEQ II) NO: 35) (SEQ ID NO: 996)
352 TTTATGTAGTTGTGTAGT
(SEQ ID NO: 35) (SEQ ID NO: 997)
353 AGTGTATGCGTAGAAGGT
SE ID NO: 35) (SEQ ID NO: 998)
354 AGTGTATGTGTAGAAGGT
(SEQ ID NO: 35) (SEQ ID NO: 999)
355 TTTAGATACGAAATGTTA
(SEQ ID NO: 35) (SEQ ID NO: 1000)
356 TTTAGATATGAAATGTTA
(SEQ ID NO: 35) (SEQ ID NO: 1001)
357 AAGTAAGTCGTTGTTGTT
(SEQ ID NO: 35) (SEQ ID NO: 1002)
358 AAGTAAGTTGTTGTTGTT
(SEQ ID NO: 35) (SEQ ID NO: 1003)
359 TTTCGTCGGAGGAATT
(SEQ ID NO: 25) (SEQ ID NO: 1004)
360 GTTTTGTTGGAGGAATT
(SEQ ID NO: 25) (SEQ ID NO: 1005)
361 ATCGTTTTGTCGGACGG
(SEQ ID NO: 25) (SEQ ID NO: 10
362 ATTGTTTTGTTGGATGG
SE ID NO: 25) (SEQ ID NO: 1007)
363 TGTCGCGATATATCGA
(SEQ ID NO: 25) (SEQ ID NO: 1008)
364 TTTGTTGTGATATATTGAT
(SEQ ID NO: 25) (SEQ ID NO: 1009)
365 AGCGTCGATTAATCGT
(SEQ ID NO: 36) (SEQ ID NO: 1010)
366 TTAAGTGTTGATTAATTGT
(SEQ ID NO: 36) (SEQ ID NO: 1011)
367 TTCGGTCGGGTTTAAA
SE ID NO: 36 SE ID NO: 1012
368 GTTTGGTTGGGTTTAAA
(SE Q ID NO: 36 SE IDNO: 1013
369 TAATCGTTAGCGGCGG
SE ID NO: 36)
(SEQ ID NO: 1014)
227
CA 02528853 2005-12-23
No: Gene O ' o:
370 TTAATTGTTAGTGGTGG
SE ID NO: 36) (SEQ ID NO: 1015
371 TTAACGGGTGGGTACGT
(SEQ ID NO: 36) (SEQ ID NO: 1016)
372 TTAATGGGTGGGTATGT
(SEQ ID NO: (SEQ ID NO: 101
373 AGGTCGTTGGTATTCGT
(SEQ ID NO: 36) (SEQ ID NO: 1018)
374 AGGTTGTTGGTATTTGT
(SEQ IDNU:3 (SEQ IDNO:1019
375 TTTTCGAGTI"TTATCGA
(SEQ ID NO: 36) (SEQ ID NO: 1020
376 TTTGAGTTTTATTGAGGT
SE ID NO: 36) SE ID NO: 1021)
377 ATAGTCGTGGfiTTCGT
(SEQ ID NO: 36) (SEQ ID NO:1022
378 ATAGTTGTGGTTTTGTT
E ID NO: 36) ID NO: 1023)
379 TGACGGGCGTTTTCGA
(SEQ ID NO: 36) (SEQ ID NO: 1024
380 GATGGGTGT'1 TTTGAG
(SEQ ID NO: 36) S8 ID NO: 1025
381 TAATGAGCGCGTTGTA,
SE IIID NO: 36) (SEQ ID NO: 102
382 ATGAGTGTGTTGTATTT
(SEQ ID NO: 36) (SgQ ID NO: 1027)
383 TTGGTTCGGGAAAGGTAA
(SEQ ID NO: 28) S ID NO: 1028)
384 TTGGTTTGGGAAA.GGTAA
(SEQ ID NO: 28) SE ID NO: 1029)
385 TTTCGGTGAATCGGAT
SE ID NO: 28) (SEQ ID NO: 1030
386 TTTTTGGTGAATTGGAT
ID NO: 28 SE ID NO: 1031)
387 TTCGTAAAGTCGTTGT
(SEQ ID NO: 28 (SEQ ID NO: 1032)
388 GGTTTTTTGTAAAGTTGT
(SEQ Il) NO: 28) (SEQ ID NO: 1033)
389 GTTTAGTTAGCGGGTTTT
(SEQ ID NO: 28) (SEQ ID NO: 1034)
390 GTTTAGTTAGTGC',GTTTT
(SEQ ID NO: 28) (SBQ ID NO: 1035)
391 GGGCGCGTACGGTTAT
SE ID NO: 28) (SBQ ID NO: 1036)
392 AGTTGGGTGTGTATGG
S ID NO: 2 SE ID NO: 103
393 TTATCGCGCGTGGAGG
S ID NO: 28) (SBQ ID NO: 1038
394 TTATTGTGTGTGGAGGA
S Il7 NO: 28) S ID NO:1039
228
CA 02528853 2005-12-23
No: Gene Oli o:
395 AAAACGTGGACGTTTT
(SgQ ID NO: 37) (SEQ ID NO: 1153)
396 ATTTGGAGCGAGGAATTT
(SEQ ID NO: 3 (SEQ ID NO: 1154)
397 ATTTGGAGTGAGGAATTT
SE ID NO: 37) (SEQ ID NO:1155
398 TTGAAAGTCGGTTAAAGT
S ID NO: 3 Sfi ID NO: 1156)
399 TTGAAAGTTGGTTAAAGT
SE ID NO: 37) (SEQ ID NO: 1157)
400 GGTAGTTACGAGAGAGTT
(SEQ ID NO: 37) (SEQ ID NO: 1158)
401 GGTAGTTATGAGAGAGTT
(SEQ ID NO: 37) S IDNO: 1159
402 GGTGCGCGTAGAGAAT
S ID NO: 26) (SEQ ID NO: 1040)
403 GGTGTGTGTAGAGAATA
(SEQ ID NO: 26) (SEQ ID NO: 1041)
404 TAAGCGTATCGACGTT
(SEQ ID NO: 26) (SEQ ID NO: 1042)
405 ATT"I"TAAGTGTATTGATGT
(SEQ ID NO: 26) (SEQ ID NO: 1043)
406 AAATATCGAACGGGAT
(SEQ ID NO: 26) (SEQ ID NO: 1044
407 ATTGAATGGGATTTAGAG
(SEQ ID NO: 26) (SEQ ID NO: 1045)
408 TTAGAGTTCGAGTTTATA
(SEQ ID NO: 26) SE ID NO: 1
409 TTAGAGTTTGAGTTTATA
SE ID NO: 26) (SEQ ID NO: 1047)
410 TTAGGCGCGGATTCGT
3E ID NO: 26) (SEQ ID NO: 1048)
411 TAGGTGTGGATTTGTT
(SEQ ID NO: 2 S ID NO: 1049
412 TTCGCGAAGTTACGGG
(SEQ ID NO: 26) (SEQ ID NO: 1050)
413 TTTGTGAAGTTATGGGT
(SEQ ID NO: 26) (SEQ ID NO: 1051
414 TATCGGTTCGGAGTTA
(SEQ ID NO: 26) SE ID NO: 1052)
415 ATTGGTTTGGAGTTAGA
SE ID NO: 26) (SEQ ID NO: 1053)
416 AAGTAGCGTCGTTATT
(SEQ ID NO: 26
(SEQ ID NO: 1054)
417 AAGTAGTGTTGTTATTGA
(SEQ ID NO: 26) SE ID NO: 1055)
418 GTCGTTCGGA.ATTCGT
(SEQ ID NO: 2 S ID NO: 1056
419 AGTTGTTTGGAATTTGT
(SEQ ID NO: 2 (SEQ ID NO: 105
229
CA 02528853 2005-12-23
No: Gene Oli o:
420 TACGTGGTCGAGGGTT
SE ID NO: 26) (SEQ ID NO: 1058
421 TATGTGGTTGAGGGTT
(SEQ ID NO: 26 (SEQ ID NO: 1059)
422 GGAAGTTTCGATGGTTTA
(SEQ ID NO: 26) (SEQ ID NO: i060
423 GGAAGTTTTGATGGTTTA
(SEQ ID NO: 26) (SEQ ID NO: 1061)
424 GGCGTTGGTATCGTTGA
(SEQ ID NO: 38) (SEQ ID NO: 1062
425 GGTGTTGGTATTGTTGA
(SEQ ID NO: 38) (SEQ ID NO: 1063
426 TTAAGACGCGTTTTTT
(SEQ ID NO: 38) (SEQ ID NO: 1064
427 AAGATGTGTT'TTTTGGA
(SEQ ID NO: 38) (SEQ ID NO: 1065)
428 T'ITTGTCGCGGGAATT
(SEQ ID NO: 38) (SEQ ID NO: 1066)
429 TTTTTGTTGTGGGAATT
(SEQ ID NO: 38) (SEQ ID NO: 1067)
430 ATACGTAGATTCGGAG
(SEQ ID NO: 38) (SEQ ID NO: 1068)
431 TATGTAGATTTGGAGGT
(SEQ IIID NO: 38) (SEQ ID NO: 1069)
432 GAAGTGGTCGTTAGTTTT
(SEQ ID NO: 39) (SEQ ID NO: 1070)
433 GAAGTGGTTGTTAGTTTIT
(SEQ ID NO: 39 (SEQ ID NO: 1071)
434 AAGGAATTCGTTTTGTAA
SE ID NO: 39 (SEQ ID NO: 1072)
435 AAGGAATTTGTTTTGTAA
(SEQ ID NO: 39) (SEQ ID NO: 1073)
436 AATGTTTTCGTGATGTTG
(SEQ ID NO: 39) (SEQ ID NO: 1074)
437 AATGTTTITGTGATGTTG
(SEQ ID NO: 39) (SIRQ ID NO: 1075)
438 TAAAACGAGGGAGCGT
SE ID NO: 39) (SEQ ID NO: 1076)
439 AAAATGAGGGAGTGTT
(SEQ ID NO: 39) (SEQ ID NO: 1077)
440 AGGAGTCGGTTTCGTA
SE II) NO: 27) (SEQ ID NO: 1078)
441 AGGAGTTGGTTTTGTA
(SEQ ID NO: 27) (SEQ ID NO: 1079
442 TAAAGCGCGGATATTT
SE ID NO: 27) (SEQ ID NO: 1080)
443 GGGTAAA.GTGTGGATA
(SEQ ID NO: 2 SE ID NO: 1081)
444 TTTGAGCGGGTATCGA
(SEQ m NO: 27) (SEQ ID NO: 1082)
230
CA 02528853 2005-12-23
No: Gene Oligo:
445 TGAGTGGGTATTGAGT
(PQ ID NO: 27) (SEQ ID NO: 1083
446 TAGAGTCGAGGGGCGG
(SEQ ID NO: 27) (SEQ ID NO: 1084
447 TAGAGTTGAGGGGTGG
SE JD NO: 27) (SEQ ID NO:108
448 TTTCGAGGGACGGAAG
(SEQ ID NO: 27) (SEQ ID NO: 1086
449 TTT"TGAGGGATGGAAG
SE ID NO: 27) (SEQ ID NO: 108
450 TATGTTTTCGGCGAAT
(SEQ ID NO: 2 (SEQ ID NO: 1088
451 TTTTGGTGAATGGGGA
(SEQ ID NO: 27) (SEQ ID NO: 1089
452 ATAGTCGAGGAGTCGT
SE IDNO: 27) (SEQ ID NO: 1090)
453 AATAGTTGAGGAGTTGT
SE ID NO: 27) (SEQ ID NO: 1091)
454 ATTTGTTTCGATTAATTT
(SEQ ID NO: 29) (SRQ ID NO: 1092)
455 ATTTGTTTTGATTAATTT
(SEQ ID NO: 29) (SgQ ID NO: 1093)
456 AATTTGCGAACGTTTGGG
(SEQ ID NO: 29) (SEQ ID NO: 1094)
457 AATTTGTGAATGTTTGGG
SE ID NO: 29 (SEQ ID NO: 1095
458 GTCGATGTTTTCGGTA
SE ID NO: 29 (SEQ ID NO: 1096
459 GGTTGATGT'ITTTGGTA
(SEQ ID NO: 29) (VEQ ID NO: 109
460 GAGTTTCGTTATATCGT
(SEQ ID NO: 31) (SEQ II) NO: 1098
461 GGAGTITTGTTATATTGT
(SEQ ID NO: 31) SE ID NO: 1099)
462 TTTTTGCGGTTCGATA
(SEQ ID NO: 31 S ID NO: 1100
463 AATTTTTGTGGTTTGATA
S ID NO:31 S IDNO:1101
464 TACGTTAAGGTTAACGTATA
S ID NO: 31 (SEQ ID NO: 1102
465 TATGTTAAGGTTAATGTATA
S ID NO: 31 (SBQ ID NO: 1103)
466 TGTTTCGTCGTTATAAT
(SEQ ID NO: 31) (SEQ ID NO: 1104)
467 GTTTTGTTGTTATAATTAGA
SE ID NO: 31 (SEQ ID NO: 110
468 GGCGTAGGTTACGATT
(SEQ ID NO: 31 (SEQ ID NO: 1 I
469 GGGGTGTAGGTTATGA
(SEQ IDNO: 31 (SEQ ID NO: 1 I
231
CA 02528853 2005-12-23
No: Crem O!' o:
470 ATTCGTTACGGATCGT
S ID NO: 31 (SEQ ID NO:1108
471 TTTATTTGTTATGGATTGT
S ID NO: 31 SE ID NO:1109
472 AGTI'iTCGGATTCGAA
(SEQ ID NO: 31 ID NO:1110
473 AGAGTTTCfGGATTTGA
(SEQ ID NO: 31 (SEQ ID NO:1111
474 TATTTCGAGGTAGCGG
SE ID NO: 31 SE ID NO: 1112)
475 TTTGAGGTAGTGGGAT
(SEQ ID NO:31 (SEQ ID NO:1113
476 GAGAGAAACGGTTTTTGT
(SEQ ID NO: 31 SE ID NO:1114
477 GAGAGAAATGGTTTTTGT
(SEQ ID NO: 31) SE ID NO: 1115
478 GTTTGATGGATGTTTTF
(SEQ ID NO: 31 SEQ ID NO: 111
479 GTACGACGGTAAGGAT
(SEQ ID NO: 31) SE ID NO: 1117)
480 GTATGATGGTAAGGATTA
SE ID NO: 31) SB ID NO: 1118
481 AGTTGTTTCGTAGATATT
(SEQ ID NO: 31 (SEQ ID NO:1119
482 AGTTGTTTTGTAGATATT
SE ID NO: 31 (SEQ ID NO: 1120
483 AGTAAGCGGTTGTATAT
(SEQ ID NO: 40) (SEQ II) NO: 1121
484 AAAAGTAAGTGGTTGTAT
S ID NO: 40 S ID NO:1122
485 AAATTGAGCGTTTATGT
SE ID NO: 40 S ID NO: 1123
486 ATTGAGTGTTTATGTGTA
S II) NO: 40 (SEQ ID NO: 1124
Table 3: Qenes or aeuornic locus associated with g~mtc seauences aocacdine to
SBO ID NO
1=S~Q ID NO 64.
Genomic. isulfite SEQ ID bank Itef. ID esariptioo.
EQ ID os: o:/contig
O=
1 04, 305, 420 & 55481,12.1.12 ypothetical protein leucine rich repeat
21 43 ega gene ID
OTTKUMG00013001328
06. 307, 422 & C02760125.1.1 azacytidine induced (Ensembl gene ID
23 7 2 1415
08, 309, 424 ~t 711.4. .88 n known gene, close lo ATF/GTP-
25 1 site motif A-loo protein _Wigg 232
CA 02528853 2005-12-23
310, 311, 426 & M_002938 ING FINGER PROTEIN 4; RNF4
27
12, 313, 428 & 021926 OMEOBOX PROTEIN ARISTALESS-
29 IKE 4;. ALX 4
314, 315, 430 & 4M_004852 NE CUT DOMAIN FAMILY MEMBER
31 ; ONC2
16, 317, 432 & M 002507 UMOR NECROSIS FACTOR
33 CEPTOR SUPERFAMILY MEMBER
16 PRECURSOR (LOW- AFFINITY
ERVE GROWTH FACTOR
ECEPTOR) (NGF RECEPTOR) (GP80-
GFR) (P75 ICD) (LOW AFFINiTY
UROTROPHIN RECEPTOR
SNTR). NGFR
118, 319, 434 & 1M 016192 TRANSMEMBRANE PROTEIN WITH
135 GF-LIKE AND TWO FOLLISTATIN-
IKE DOMAINS 2;
TRANSMEMBRANE PROTEIN TENB2;
T'OMOREGULIN; PUTATIVE
TRANSMEMBRANE PROTEIN WITH
GF-LIKE AND TWO FOLLISTATIN-
IKE DOMAINS 2.
20, 321, 436 & 4M_005221 OMEOBOX PROTEIN DLX-5
37
122, 323, 438 & 4M_080552 ESICULAR INHIBITORY AMINO
139 CID TRANSPORTER (GABA AND
LYCINE TRANSPORTER)
VESICULAR GABA TRANSPORTER)
HVIAAT)
11 124, 325, 440 & M_001208 ranscription factor BTF3 Homolog 1
441
12 26, 327, 442 & M_005904 OTHERS AGAINST
43 ECAPENTAPLEGIC HOMOLOG 7
SMAD 7) (MOTHERS AGAINST DPP
OMOLOG 7) (SMAD7) (HSMAD7).
13 328, 329, 444 & _002146 OMEOBOX PROTEIN HOX-B3 (HOX-
5 G (HOX-2.7)
14 330, 331, 446 & M 025078 Homo sapiens hypothetical protein
447 FLJ22378
332, 333, 448 & 001116 [ADENYLATE CYCLASE, TYPE IX (EC
449 1.6.1.1) (ATP PYROPHOSPHATE-
YASE) (ADENYLYL CYCLASE).
DCY9
16 334, 335, 450 & 4M_001706 3-CELL LYMPHOMA 6 PROTEIN
151 BCL-6) (ZINC FINGER PROTEIN 51)
LAZ-3 PROTEIN) (BCL-5).
17 436,337,452 &
53
18 338, 339, 454 & _017745 CL-6 INTERACTING COREPRESSOR
55 SOFORM 1 BCOR
233
CA 02528853 2005-12-23
55 OFORM 1 BCOR
19 340,341,456& 005643 SCRIPTION INFTIATION
57 ACTOR TFIII3 28 KDA SUBUNIT
AFII 28) (TAFII28) (TFIID SUBUNIT
30-BETA
d 42, 343, 458 & _007374 OMEOBOX PROTEIN SIX6 (SINE
59 HOMEOBOX HOMOLOG 6)
OPTIC HOMEOBOX 2)
OMEODOMAIN PROTEIN OPTX2).
1 44, 345, 460 & 3 500.2.1.16 itusted between Ensemble gene IDs
461 8 N L%TGM23086d9 and
S Igoooizvgfig
22 46, 347, 462 & C 385.4. .97 PIR-2 PROTEIN (FRAGMENT)
463 18
3 48, 349, 464 & C 7 223.5.1.15 itusted witkin Ensemble EST ID
52 ESTGd0001971415
4 150, 351,466 & 005229 TS-DOMAIN PROTEIN ELK-1
7
5 52, 353, 468 & 000179 NA MISMATCH REPAIR PROTEIN
169 H6 (MUTS-ALPHA 160 KDA
UBUNIT) (G/T MISMATCH BINDING
ROT (GTBP) GTMBP (P160)
6 54, 355, 470 & 005427 OR PROTEIN P73 (P53-LIKE
71 CRIPTION FACTOR) (P53-
TED PROTE .
7 156, 357, 472 & _002093 LYCOGEN SYNTHASE KINASE-3
?3 ETA (EC 2.7.1.37 GSR-3 BETA).
158, 359, 474 & 006765 33 PROTEIlV'
9 60, 361, 476 & _016192 MEMBRANE PROTEIN WTTH
177 GF LIKE AND TWO FOLLISTATIN-
IKE DOMAINS 2;
MEMBRANE PROTEIN TENB2;
OMOREGULIN; PUTATIVE
sMEMBR-ANE PROTEIN WITH
GF-L1KE AND TWO FOLLISTATIN-
DOMAINS 2.
10 62, 363, 478 & 004429 HR1N B1 PRECURSOR (EPH-
79 TED RECEPTOR TYROSINE
ASE LIGAND 2) (LERR-2) (ELK
IGAND (ELK-L).
1 364,365,480 & _018950 -F gene for human leukocyte antigen
481
2 66, 367, 482 & 000038 ENOMATOUS POLYPOSIS COLI
493 ROTEIN (APC PROT
3 368, 369, 484 & 000610 ANTIGEN PRECUR.SOR
185 HAGOCYTIC GLYCOPROTEIN 1)
GP-1) (HUTCH-I)
CELLUL.AR. MATRIX
CEFTOR- (ECMR-III) GP90
234
CA 02528853 2005-12-23
YMPHOCYTE HOMING/ADHESION
E1'TOR) (HERMES ANTIGEN)
ALURONATfi RECEPTOR)
ARAN SULFATE
ROTEOGLYC (EPICAN) CDW44
4 70, 371, 486 8t 004385 VERSICAN CORE PROTEIN
487 RECURSOR (I.ARGE FIBROBLAST
ROTEOGLYCAN) (CHONI7ROITIN
ULFATE PROTEOGLYCAN CORE
ROTEIN 2) (PG-iVi) (GLIAL
ALURONATE-BIlNDING PROTEIN)
GHAP CSPG2
35 [74,375,490& 2, 373, 488 & A 4 EnsEMBL ID ENSG00000112319
9
6 000455 ERINE/THIZEONINE-PROTEIIJ
1 E l 1(EC 2.7.1.-)
SERINF./TFIl~EONINE- PROTEIN
ASE LKBI
I
7 76, 377, 492 & 003242 raasfomiing growth factor, beta receptor
93 11 GFBR2
8 178, 379, 494 & 001753 AVEOLIN-1; CAV 1
9 80, 381, 496 & 001257 HERIN-13 PRECURSOR
97 UNCATED-CADHERIN) (T-
ADHFRiN) (T-CAD) (HEART-
ADH -CAD 105
40 82, 383, 498 & 000044 ROGEN RECEPTOR
99 IHYDROTESTOSTERONE
CBPTOR AR
1 84, 385, 500 dt 032546 G FINGER PROTEIN 30, RNF 30,,
501
2 386, 387, 502 & 033178 UBLE HOMEOBOX, 4; DOUBLE
503 OMEOBOX PROTEIN 4. DUX4
3 388, 389, 504 & _003573 TENT TRANSFORM[NG GROWTH
505 ACTOR BETA BINDING PROTEIN 4.;
TBP4
90,391, 506 & GI-20 PROTEIN
07
5 392, 393, 508 & 014459 ROTOCADHERIN 17;
509 ROTOCADHERIN 68.; PCDH 17
46 94, 395, 510 & 005285 ROBABLE G PROTEIN-COUPLED
511 EPTOR GPR7.; GPR7
17 396, 397, 512 & _016269 YMPHOID ENHANCER BIND1NG
513 ACTOR l (LEF-1) (T CELL-SPECIFIC
SCRIPTION FACTOR 1 ALPHA)
CF1-ALPHA
48 98, 399, 514 & AC139426.2-1,18 ituated between F.ngRhMID
15 908 S TGQ 3302Q72 and EnsEMBL ID
33
9 , 401, 516 & 005904 OTHERS AGATNST
235
CA 02528853 2005-12-23
517 ECAPENTAPLEGiC HOMOLOG 7
SMAD 7) (MOTHERS AGAINST DPP
OMOLOG 7) (SMAD7) SMAD .
0 402,403,518 & 12590.2.1.165 omo sapiens mRNA for KIAA1529
19 32 rotein EnsEMBL ID ENSG00000029402
51 104, 405, 520 & _001866 OX7B
21
lAAA
2
, 407, 522 & 001900 ST5
23
3 108, 409, 524 & 001990 YES ABSENT HOMOLOG 3. EYA3
4 10, 411, 526 & 002032 TIN HEAVY CHAIN (FIy.RRITIN
27 SUBUNI . FTH1.
55 12, 413, 528 & _002938 G FINGER PROTEIN 4.; RNF4
29
6 14, 415, 530 & PI EnsEMBL ID ENSG00000144339
31
7 116, 417, 532 & _000852 LUTATHIONE S-TRANSFERASE P
533 C 2.5.1.18) (GST CLASS-PI) (GSTP1-
1
8 18, 419, 534 & _007084 RANSCRIPTION FACTOR SOX-21
9 5, 66, 77 & 78 003299
0 7, 6$ 79 & 80 025218
I 9, 70, 81 & 82 INM 001374
2 1, 72, 83 & 84 003714
3 3 74,85 & 86 002196
5, 76, 87 & 88 000484
236
CA 02528853 2005-12-23
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