Note: Descriptions are shown in the official language in which they were submitted.
f.
CA 02369183 2002-O1-23
Title: Means and methods for treatment evaluation.
The invention relates to the field of u~edicine. The invention
paxticularly relates to the fields of xnaleeular biology and detection
methods.
Recent advances in the lrnov~rledge of molecular processes in a cell
and techniques to study these processes have resulted in improved methods of
typing and treating diseases. Understanding of the underlying molecular
diversity of tumors has, for instance, already led to a better txx~derstanding
of
the diversity of response to treatment of morphologically similar tumors.
Tmproved typing influences the way tumor patients are being treated. A
drawback of the current methods of treatment is, however, that it takes a
relatively long time to determine whether a treatment given to a patient is
actually effective. rfhis impedes the optimization of dosages and/or schedules
with which treatment is given. Moreover, it also slows down the possibility to
adjust the txeatment regimen all together. For instance, adjustment of
therapy is currently only possible ~rhen macroscopic arxalysis of tumor cells
in
the body indicates that the therapy given is not effective. Macroscopic
changes
typically need several weeks to manifest themselves and equipment to
meas~zxe such changes is often not readily available.
The present invention provides a method for determining whether a
24 treatment is effective in changing a status of a certain set of target
cells in an
individual comprising obtaining a sample froze said individual after
initiation
of said treatment and detsrminzng whether said sample comprises an
expression product of at least one marker gene. In one embodiment of the
invention said set of target cells coxrlgxises a tumor cell. By changing a
status
2~ of a set of target cells is zrieaxxt herein that at least one property of
said set of
target calls is altered. For instance, the amourxt of said target cells may be
changed. Said amount may either ~be increased or decreased. Alternatively, the
activi of said target cells may be,altered. Said activity may be a replication
d
CA 02369183 2002-O1-23
z
activity. As another example, said activity rnay be an activity involved with
angiogenesis. Alternatively, said activity may be an apoptatic activity.
It was found that tuznox cells andior surrounding tissue respond, on
a molecular level, very quickly to an effective treatment. This response can
be
detected by measuring an expression product of a marker gene. I4farker gene
expression products are indicative for a response to treatment. l~2arker genes
are typically genes that are expressed by said set of target cells, for
insta.x~ce
tumor cells, and/or surrounding tissue. However, marker genes can also be
expressed in non-tumor target cells in other compartments of the body, for
1Cl instance blood cells andlor cardiovascular cells.
Alternatively, marker gene e~cpression can be initiated upon
treatment given to the individual. Marker gene e~tpxession products are
responsive to treatment given to a patient. A response caxk be an alteration
1n
the relative amounts o~ expression product. However, it can also be an
15 alteration i~a. absolute presence or absence of expressed product such as
R3~TA
and/or protein.
Accordir~g to the invention, a sample which is obtained from a
patient may comprise at Ieast one of said target cells. fihis is particularly
suitable fax detecting circulating tumor cells which have released tlxemselves
20 from a tumor and are circulating in the blood of a patient. Alternatively,
said
target cells may be non-tumor cells: In another embodi.me~t of the invention,
said sample does n.ot comprise any target cello. Ho~ewer, said sample may
comprise another, non-target cell. Expression, or change of expression, of at
least one marker gene by said non-target cell is indicative for the status of
a
25 certain set of target cells. Said non-target cell preferably camprises a
peripheral blood mononuclear cell, as is described below. In yet another
embodiment, said sample does not comprise any cell at aLl. For instance, no.
expression product of a marker gene, produced by a. target cell or non-target
cell else~avhere in an individual's body, may be present in said sample at
3o detectable levels,
y.
CA 02369183 2002-O1-23
3
With a method of the invention it is possible to determine whether a
treatment is effective in said individual. This can be done whi~.e a treatment
is
given or shortly after said treatment. Thus it is possible for instance to
adjust
treatment schedule, dosages and type on a patient per patient basis. It is
preferred that said sample is obtained within a week of initiation of
treatment.
More preferably, said sample is obtained ~ovithixx two days of initiation of
treatment. 'Vfith a method of the invention it is possible to evaluate
treatment
effectiveness almost immediately after initiation of said treatment. A method
of the invention thus offers a good opportunity for determining whether
to treatment adjustments are required.
A marker gene preferably comprises a gene involved in the
gexseration, maintenance and/or breakdown of blood vessels (angiogenesis),
Classes of genes involved ixi the process of angiogenesis encompass among
others receptors, ligands and signaling molecules. Tumor cells are dependent
15 on the growth of new blood vessels to maintain expansion of turxtor mass.
On
the one hand, blood vessels are required to carry nutrierets to the site of
the
tumor, whereas on the other hand waste material needs to be transported from
the tumor. In the present invention it has been shown that expression
products from genes involved in the generation, maintenance and breakdown
20 ox a blood vessel are among the ~'trst to respond to anti-tu~.or
treatments. Such.
genes are therefore very suitable marker genes of the invention. In ox~e
embodiment said marker gene comprises a sequence as depicted in table 1. or
2. In another embodinZent said marker gene comprises a sequence as depicted
in ftguxes 1-1.8, or a part or analogue thereof. In a preferred embodiment
said
~5 marker gene comprises a TIE 1 sequence, a Salioadhesin or Siglec 1
seque~xce,
a sequence as depicted in. ~.gure $ or 17, or a part or analogue thereof.
A change in the level of expression product of a marker gene is
indicative far whether a treatment is effective or not. For instance, the
level of
espressian product of a marker gene can be enhanced. in a sau~ple when a
30 treatment is effective, alternatively expression product of a marker gene
ca~x
f
CA 02369183 2002-O1-23
be reduced. Thus, preferably, e~epression product of a marker gene is
quantified. The level of expression product in a sample can vary due to
changes in the expression of a marker gene. ~Towever, it is also possible that
the level changes due to a change in type of cells comprising said expression
product in said sample, for instaxace due to treatment related cell death at
the
site of the body where the sample is obtained. Cbnsidering that the level of
expression product of marker genes can vary ~rom patient to patient, it is
preferred that a xaethod of the invention further comprises comparing the
level
of expressio.tt product of said masker gene with a references 1'referabiy said
reference comprises the same type of tumor cells prior to, or in the absence
of,
said treatment. Preferably, said tumor cells are derived from the same
patient.
The difference in the level of expression product of a mar);er gene ixt an
effective and a non-effective treatment can be very large. rn the extreme
cases
the level of expresszan product can range from detectable to not detectable.
1a Il~~arker genes displaying such zero to one relation in expression product
levels
are preferred in the present invention. A zero to one relation can be used to
design relatively simple test systems. A zero t;o one relation, is of course
dependent on the detection system used to detect expression product of a
marker gene. Very sensitive expression detection systems will typically detect
expression product where a less sensitive syaterns detects no expression
product. An expressioxl product can be RNA or a part thereof, transcribed from
said marker gene or a translated protein or a part thereof. A person skihed
ixi
the art' is yell capable of designing the most appropriate expression
detection
system to practice this preferred embodiment of the invention.
A part of an RNA or NINA molecule is defined herein as an RNA or
IaNA sequence, copzprising at least 50 nucleot,-~.des. A part andlor au
analogue
of an expression product is defined herein as a part and/or analogue that can
be detected using essentdally the same kind of detection method as said
expression product, altriough the sensibility of detection may differ. An
analogue of an RNA ox DNA molecule is de$.ned herein as an RNA or 1.~N'A
f 1
CA 02369183 2002-O1-23
sequence which is essentially the same as a particular RNA or DNA sequence.
However, a 'nucleotide mutation, replacement, alteration, addition andlor
deletion. may have taken place naturally and/or performed artificially,
without
essentially altering the detection of said analogue as compared with the
detection of said particular RNA ox DNA sequence. A person skilled in the art
is well able to dexermine whether a given RNA or TINA sequence is an
analogue of a particular RNA or T3NA sequence, using techniques known in
the art.
In a preferred embodiment said tumor cotuprises Ii.a,posi's Sarcoma.
Kaposi's Sarcoma is a disease of proliferating blood vessels and therefore
very
much suited for identifying rreaxker genes involved in angiogen.esis.
According
to the inventio~t,, changes in angiogenesis factors are among the first marker
eve~xts as a result of treatment. Kaposi's Sarcoma (~S) manifests itself
clinically by reddish skin lesions. haposi's Sarcoma is a multicentric,
malignant neoplastic vascular pzoliferation characterized by the development
of bluish-red cutaneous nodules, usually on the lower e:ctremities, most often
on the toes or feet, and sloavly increasing in sire ab.d number and spreading
to
more proximal areas. The tumors have endothelium-lined chancels and
vascular spaces admixed with variably sized aggregates of spindle-shaped
cells, and afters remain confined to the skin a-zxd subcutaneous tissue, but
widespread visceral involvement may occur. T~aposi's Sarcoma occurs
spontaneously in Jewish and Italian males in Europe and the United States.
An aggressive variant in young children is endemic in some areas of Africa. A
third form occurs in about 0.04% of kidney transplant patients. There is also
a
2~ high incidence in AIDS patients. (1i rom Dorland, 27th ed ~ Holland et al.,
Cancer Medicine, 3d ed, pp210~-7)
Raposi's Sarcoma is aggressive in HlC'~ infected individuals. The
angiogenic mechanism causing the lesions results from the interplay of viral
and cellular gene expression and is poorly understood in terms as to which
3o genes axe in~rolved and what controls their expression. The angiogenic
CA 02369183 2002-O1-23
6
proliferatio~z in KS involves mechanisms likely to be universal in
angiogenesis.
The central role of angiogenesis in Kaposi's Sarcoma is clearly illustrated by
the French xiame for this tumor: angiasarcontatose kaposi. Because of said
central role of angiogexxesis in Iiaposi's Saxcoma; determination of marker
genes involved in angiogenesis is very suitable to determine whether a
treatment of Tfaposi's Sarcoma is effective.
La the pxesex~t invention, gene expression patterns of ~aposi's
Sarcoma wexe examined with a method caned serial analysis of gene
I0 expression (SAGE) ("t~relculescu et al. (1995) Science 270; X84-48?). This
method allo~sws the quantitative and simultaneous analysis of a large number
of transcripts. SAGE is based on two principles. First, a short nucieatide
sequence TACT (14 base pairs) contains su~cien.t information to uniquely
identify a transcript, provided it is isolated from a defiried position within
the
I5 transcript. Second, concatenation of short sequence TAG's allows the
ef$.cient
axtalysis of transcript ii'1 a sexial z~xannex by sequencing of multiple TAG's
within a sixigle clone.
Briefly, in this method a biotinylated oligo (dT) primer is used to
synthesize eDNA from mI~NA, and after digestion with a restriction enzyme,
20 the most 3' terminus (near the poly-A tail) is Rsol~ted. These 3'
fragxrxents of
cDNA are ligated to linkers and cleaved with a type TI restriction enzyme to
release short sequence (14 bp) of the original cDNA {TAG's). The TAG's are
ligated to diTAG's and pCR ampli$ed. These di-TAG's are then ligated to form
long concatamers, which are cloned and sequenced. In this way, one seq~xence
25 reaction yields information about the distribution of raany different
mRNA's.
Finally, the ca.lculataor~ of the abundance of different TAG's and the
xnatehing
of the TAG's in Genbanlt are dox~,e using the necessary computer software.
Xn another aspect the invention provides the use of a nucleic acid
comprisi.x~g a sequence as dspicxed in f gure 1-18 audlor table 1 or 2, or a
part
30 or analogue thereof, in axe expression product detection method. Preferably
ti 1
CA 02369183 2002-O1-23
said nucleic acid comprises a TIE 1 sequence, a Salioadhesin or Siglec 1
sequence, a sequence as depicted in figure 8 or 1?, or a part or analogue
thereof: Expression of a marker gene in arr individual can be detected by
determining whether said nucleic acid or part or analogue is able to hybridize
with nucleic acid, preferably RNA, in a sample of said individual. If
hybridisation takes place, it is indicative of expression of a marker gene in
said
individual. Of course, as is knoc~rm by a person skilled in the art, a coding
strand of DN9lI~NA is capable of hybridizing with the camplexnentary strand
of a corresponding doublestranded nucleic acid sequence. Hence, a
complementary straxad of a certain coding stra~ad is particularly suitable for
detection of expression. of said coding strand. F'or instance, a complementary
strand of a coding strand as depicted in figure 1-18 andlor table 7. or 2 is
suitable fax detection of expression of a gene comprising said coding strand.
In yet another aspect, the invention provides the use of a
proteinaceous molecule capable of specifically binding a protein encoded by a
nucleic acid comprising .a sequence as depicted in figure 1-18 andlor table 1
or
2, or apart ar analogue thereof, in a detection method. Preferably, said
proteinaceous molecule is capable of specifically binding a protein encoded by
a
nucleic acid comprising a T~ 7. sequence, a Salioadhesin or SiDlec 1 sequence,
24 a sequence as depicted in figure 8 or 1?, or a part or analogue thereof. In
one
embodiment of the invention, said uses are directed tovvard determining the
presence of a site of angiogenesis in an indi iduai. In another embodiment of
the in~rention, said uses are directed toward determining the presence of a
tumor cell in an individual. The presence of a tumor cell in arr individual
care
be determined because said tumor cell typically expresses marker genes that
can be detected by an expression product detection xt~ethod. ~'or instance, an
aiatibody, or analogue thereof, specifzeally directed against an e~rpression
product of said marker gene can be generated. Said antibody or analogue is
suitable for deterxninatiou of an expression product of said marker gene in a
sample, To determine the presence of a turaar cell in au individual, a sample
9
CA 02369183 2002-O1-23
from said individual can be incubated with said antibody: If said sample
contains an expression product of said marker gene, said antibody will bind.
Binding can be demonstrated by techniques known in the art, like for instance
ELIS 4. If binding of said antibody is demonstrated, one ca.n conclude that
said
3 sample co~xtains an expression. product of said marker molecule. The
presence
of an expression product of said maxker molecule can indicate the presence of
a
tumor cell in an individual, since said marker molecule is expressed by tumor
cells. There are of course many more alternative techniques to detect an
expression product with use of a proteinaceous binding molecule, which are
well known in the axt and need no further discussion hexe. Thus, proteins
expressed by a tumor cell can be detected by a proteinaceous molecule capable
of specifically binding a protein encoded by a nucleic acid comprising a
sequence as depicted in figure 1-7.$ andlor table 1 or 2, like for instance a
TIE
1 sequence, a Salioadhesin andlor Siglee 1 sedue~ce, or a part or analogue
z5 thereof. Like~cvise, the presence of a site of angiogenesis in an
individual can be
determined by detecting an expression product of a marker gene.
In another embodiment, a use of a nucleic acid cou~.prising a
sequence as depicted in figure L-18 and/or table I or 2, or a use of a
proteinaeeous molecule capable of specifically binding a protein encoded by a
2~ nucleic acid comprising a sequence as depicted in figure 1-18 andlor table
I or
2, or a part or analogue thereof, are directed toward detexmining whether a
treatment is e~'fective in changing the status of a certain set of target
cells in
as individual. In a preferred embodiment said nucleic acid comprises a TIE 1
sequence, a Salioadhesin or Siglec 1 sequence, a sequence as depicted in
~gtxre
2~ 8 or 17, or a part or analogue thereof. In another preferred embodiment
said
proteinaceous molecule is capable of specifseally binding a protean encoded by
a
nucleic acid comprising a TIE 3 sequence, a Salioa.dhesin or Siglec I
sequence,
a sequence as depicted in figuxe 8 or I7, or a part or analogue thereof.
II~Iore
preferably, said uses are directed toward detexmi.ning whether a treatment is
CA 02369183 2002-O1-23
9
effective in counteracting a tumor in an individual. ha one embodiment of the
invention, said tumor comprises Kaposi's Sarcoma.
rn one aspect the invention provides the use of a nucleic acid
corapri3ing a sequence as depicted in table I or 2 andlor figure i-1$ as an
indicator for angiogenesis. Tn a preferred embodiment the invention provides
the use of a nucleic acid cozxtprising a T'TE 1 sequence, a Salioadhesin ox
Siglec
1 sequence, a sequence as depicted in figure 8 or 17, or a part or analogue
thereof as an. indicator for angiogenesis. For ii~.stance, a rxucleic acid
comprising a sequence as depicted in figure 1-18 andlor table ~ or 2 can be
used as detection marker for the process of angiogenesis in the course of
regenerative treatxxxent. Changes in the expression level of the detection
marker indicate active growth of blood vessels (i.e. angiogenesis) as was
meax~t
to induce with the regenerative treatment course. In a preferred embodiment
such application is in the field of heart and coronary disease sixaed at
generation of new blood supply to affected organs by means of new blood
vessels. Likewise, the treatment of tumors with azxti-angiogenesis drugs can
be
monitored by changes in expression levels of detection marker genes as
depicted in figure 7.-18 and/or table 1 or 2, such as a TIE 1 sequence, a
2o Salioadhesin andlor Siglec 1 sequence,
In another aspect the invention provides the use of a nucleic acid
comprising a sequence as depicted in figure 7.-18 and/or table 1 or 2 as
detection marker for tumor cells. In yet another aspect the invention provides
the use of a pxoteinaceous molecule encoded by a nucleic acid comprising a
sequence as depicted in figure 1-1$ and/or table 1 or 2 or a protei.t~.aceous
molecule capable of binding a protein encoded by a nucleic acid comprising a
sequence as depicted in ~.gvtre 1-18 and/or table 1 ox 2 as detection marker
for
tumor cells.
CA 02369183 2002-O1-23
GVith a method of the inventiox~ it is possible to monitor a specific
status of an individual. The presence of a disease - or danger of developing
one
- can be determined by determining whether or not a sample of an individual
comprises an expression product of a marker gene. This means that also the
absence of a maxker gene in a sample can be indicative for the presence of a
disease, or for danger of developing a disease. This is possible far any
disease,
as long as the disease involves an altered expression pattern of at least one
marker gsx~e. Preferably the presence of a nx.arker gene in a sample is
determined.
Additionally, a healing process can be followed as vv'ell. For instance,
recovery
of damaged tissue can involve an increasing amount of expression product of a
marker gene over time. It is however also possible that xecovery of damaged
tissue involves a deerea.sine amount of expression product of a uzarker gene
over time. Samples taken at different time intervals pr4~i.de information
about
la tha amount of expression product which is generated at different time
points.
An altered amount of a specific expression product found in samples during a
period of time is indicative of the amount of tissue cells generated.
Likewise, a
decreasing amount of an e~cpression product found in samples in a specific
time-period can indicate a certain - either benef vial ox harmful - process.
For
2~ instance, said process may involve the development or the treatment of
disease. An important application is a txeatme:nt of heart and coronary
disease. A method of the invention is very suitable for monitoring the
genexation of never cardiac tissue.
25 Thus> one aspect of the invention provides a method of diagnosis, in.
particular a method for determining whether an individual comprises a tumor
cell and/ox & site of angiogenesis, comprising obtaining a sample from an
individual, and determinixag whether said sample comprises an expression
product of at least one marker gene. Preferably, said xzxexkex gene comprises
a
30 sequence as depicted in table 1 or 2 and/or f'~.gure L-18, ox a. part or
analogue
CA 02369183 20021-O1-23
11
thereof. ll~iore preferably said marker gene comprises a TIL ~. sequence, a
Salioadhesin ox Siglec l sequence, a sequence as depicted in fiure 8 or 17, or
a
part or analogue thereof. 'With a method of the invention it is possible to
detect
tumor cells that have released themselves froxu the tuzoor and are elsewhere
in the body. In a preferred embodiment such detection is performed in the
blood of a person detecting circulating tumor cells. These circulataug tumor
cells can be used for primary identification of presence of a tumor somewhere
in the body and also for identification of the risk of metastasis of the tumor
to
other places ixx the body nest to the primary location of the body. Likewise,
a
method of the invention is suitable for determining a site of augiogenesis in
an
individual. Angiogenesis is an indicator for different aspaacts. ~'or
instance, an
increased level of angiogenesis indicates the presence of tumor cells, or the
healing of damaged tissue, Iike for instance recovery of heart and coronary
disease.
Sinee an arxgiogenic process is now easily monitored lay a method of
the invention, it is likewise easy to determine whether a certain treatrnezet
is
effective in alterixtg an angiagenic process. Fox instance, if a certain
treatment
is effective in counteracting an angiogenic process, the amount of an
expression product of a marker gene involved m angiogenesis decreases as well
oven time. In the art, many drugs axe known for anti-angiogeni.c treatment.
Thus, one embodiment of the invention provides a method for determining
whether a treatment is effective in altering an angiogenic process in an
individual comprising obtaining a sample from said individual after initiation
of said treatment, and deterxtaining whether said sample comprises an
expression product of at least oz~e marker gene.1'xefexably, said marker gene
comprises a sequence as depicted in table 1> 2, axxdlor figures 1-1.8, or a
part or
analogue thereof. Il~Iore preferably, said marker gene comprises a TIE Z
sequence, a Salioadhesin or Siglec ~ sequence, a sequence as depicted in
figure
8 or 1?, or a part ox analogue thereof. Ire one em.bodime~tt, said treatment
3o comprises counteracting ar~giogerresis in said individual: In yet another
, ._
CA 02369183 2002-O1-23
~z
embodiment said treatment involves the use of at least one of the following
drugs: 2ME2, Angiostatzn, Anaiozyme, Anti-V~GF R.huMAb, Apra (CT-2584},
Avicine, Benefm., BD1S275291, Carboxyamidotriazole, CC4047, CCa013,
CC7085, CDC801, CGP-4121 (PKG 412), CM101, Combretastatin A-4
Prodrug, EItJID I21.9?4, Endostatin, Flavopiridol, Genisteiu (GCf), Green
'T'ea
Extract, 111~I-862, >-mmTher, Interferon alpha, Tnterleukin-12, Iressa
(ZDI839),
Marimastat, Metastat (Col-3), Neovastat, Octreotide, Paclitaxel,
Penicillamine, Photofrin, Photopoint, PI-88, Prinomastat (AG-3340), PTK787
(ZFi22584), RO317453, Soli~nastat, Squalamine, SU I01, SU 5418, SU-X668,
l.0 Suradista (1~'CE 2644), Suramlu (Metaret), Tetrathiomolybdate,
Thalidomide,
TI~I'-470, and/or Vitamin. However, the artisan can think of more drugs that
can be used during said treatment.
In a preferred embodiment, a sample of a method of the invention is
a blood sauZple. Although the location of for i.x~stance an angiogerxic
process can
be a tumor or a part of the skin, a blood sample is preferred, among other
thixxgs because it is much easier to obtain. A blood sample is also often
easier
to investigate, requiring less expensive andlor specific equipment. quite
surprisix~gly, we have found that the e~epression of certain marker genes by
heraopoietic cells, like peripheral blood mononuclear cells (PB1V.1C), can be
indicative for a process occurring somewhere else in an individual's body. For
instance, the presence, or alteration in a:cnount, of au expression product of
a
marker molecule in PBMC can indicate the presence of a tumor somewhere in
the body. rn example IO it is for instance shown. that a TIE 2 aeduence (tag
15,
table 3) or a Salioadhesin or Siglec 1 sequence (tag 3~, table 4) are both
upxegulated in skin tumor and in PBMC cells in a Kaposi's Sarcoma patient.
Additionaiy, example 8 shows that the absence of expression product of a
Kexatin 14 sequence (tag 7, table 3) in a blood sample of said patient,
whereas
Kexatiu 14 is overespressed in tumox cells, indicates that said sample was not
contaminated with tumor cells. Likewise, the e5cpressiorx of certain ui.arker
CA 02369183 2002-O1-23
13
genes by P811dC can provide another diagn.oatic indication. The presence or
absence of an expression product of a marker ge~.e in P13MC provides adequate
information about di~~ereut aspects andlor processes of an individual's body.
Preferably, the amount of expression product in a non-hemopoieteic cell is
compared ~rith a refereu.ce value. This way an indicatioze is obtained about
an
increment or decrement of e:fpressian in said hexnopaietic cell.
In one aspect the invention therefore provides a method far
determining whether an individual comprises a nor-hemopoietic tumor cell
arcdlar a site of angiogerlesis, said method comprising determining whether a
hemQpoietic cell from said patient comprises au altered amount of an
expression product of a marker gene as compared with a reference wafue.
Preferably, said marker gene coxaprises a gene involved in angiogenesis. Mare
prefErably, said gene comprises a sequence as depicted in table 1 or 2, andlor
figures 1-18, or a part or analogue thereof. Most preferably, said gene
comprises a TIE 1 sequence, a Salioadhesin or,Siglec 1 sequence, a sequence as
depicted in figure 8 or 17, or a part or analogue thereof. In one embodiment
of
the invention said hemopoietic cell comprises a peripheral 'blood xnononucleax
cell.
?o In one aspect the invention provides a method of the invention,
wherein said expressiol~. product is expressed by a PBNIC. Preferably, said
expression product comprises a TIE 1 sequence, a Salioadhesin or Siglec 1.
seque~lce, a sequence as depicted in figure 8 ox 17, or a part or analogue
thereof. As these sequences are involved in angiogenesis, the invention also
provides a use of a PBMC expressed Keratin 14 sequence, TIE 1 sequence,
Salioadhesin or Siglec 1 sequence, a sequence as depicted in figure 2, 8 or
L7,
or a part or analogue thereof, as an indicator far angiogenesis. Li$erx~ise,
these
sequences are involved in the presence of tumor cells. Therefore, a use of a
pl3riiC expressed Keratin. ~.~4 sequence, TIE 1 sequence, Salioadhesin or
Siglec
1 sequsuce, a sequence as depicted in h.gure 2, 8; or 17, or a part or
analogue
CA 02369183 2002-O1-23
1~
thereof, for determining the presence of a tumor cell in an ixidx~~idual, is
also
herewith provided. Additionally, the i.rwention also provides an isolated
keratin 14 sequence, TIE 1 sequence, Salioadhesin or Siglee 1 sequence, a
sequence as depicted in figure 2, 8; or 17, or a part or analogue thereof, for
use
in a diagnostic method. A diagnostic method can be carried out using a
diagnostic kit. Therefore, a diagnostic kit comprising a nucleic acid
comprising
a sequence as depicted in figure 1-1$ anafor table 1 or 2, or a part ox
analogue
thereof, andlox a proteinaceous molecule capable of specifiically binding a
protein encoded by said nucleic acid or said part or analogue, is also
herewith
provided. Preferably, said kit comprises a suitable mEans of detection. In one
embodiment a diagnostic kit of the invention is provided comprising a
T~erati.n
14 sequence, andlor a TIE 1 sequence, andJor a Sahoadhesin ox Siglec 1
sequence, andlor a sequence as depicted in figure 2> 8 or 17, or a part ax
analogue thereof.
A diagnostic lot of the invention is particulaxly useful for carrying out a
method of the invention. In yet another embodiment the inventiox~ therefore
provides a use of a diagnostic kit of the invention for determining whether a
treatment is effective in changing the status of a cextain set of target cells
in
an .individual andlox altering an angiogenic pr. ocess in an individual.
~dditionaliy, the invention provides a use of a diagnostic kit of the
invention
far determining whether an individual comprises a tumor cell andlor a site of
angiogenesis.
With a marker gene of the invention it is possible to screen for drugs
dixected against a disease for which said marlter gene is indicative. There
are
many methods available in the axt for screening for a speci.~.c drug activity.
For instance, eaa.le oan ire incubated with different ,potential drug
compounds,
and an expression pattern of a znarkex gene in said cells before and after
exposure to each potential drug compound can be compared. A specific
diffexence ix~ an erpression patter~x after exposure to a particular potential
3~ drug compound shows that said compound is a suitable candidate for the
CA 02369183 2002-O1-23
~~J
development of a medicament. The in ention therefore provides in one
embodi~n.ent a use of an e:~pression product of a gene coz~prising a sequence
as
depicted in figure 1-18, table 1 or table 2 as a drugtarget_ Preferably, said
sequence is a Salioadhesin or siglec 1, TTE 1, andlor Keratin 14 sequence. A
compound capable of altering the activity of Salioadhesin or SigIec 3, TiE 7.,
Keratin 14, andlor the expression of Salioadhesin ox Siglec 1, TIE 1, andlor
Keratin 14 in a cell is also herewith provzded. Said compouMd is garticularly
suitable for the preparation of a medicament.
~0
The present invention is further explained i.t~ more detail by the
follo~cv~ing examples, which do not limit the inve~.tion ixt any way.
CA 02369183 2002-O1-23
16
Examples
hJxanzple 1
Tn this e~cample a selection of samples for analysis of expression profiles is
made.
A 31-year old man was demonstrated to be HI'~7~-1 seropositive in 1i ebruary
1997. The inita.al C174 cell count was 25 x 146/ 1. The patient presented
within
two months a mucocutanous Herpes simplex infection and an extrapulmonary
Cxyptococcosis far which sp2ci~.c medication was given. The H1V-1 RNA load
at presentation was 1~,(~00 copies/ml and increased to 83,000 copiesfml in
three months. Then antiretroviral therapy was started with ri.dovudine,
lamivudine and indanavix, immediately after start therapy the HIV'-1. RNA
load dropped below detection limit. In November 1997 the patient presented
with gradual appearance of au increasing nuxriber of violaceous skin lesio~xs
that clinically resembled K.aposi's Sarcoma. The diagnosis was confirmed by
histological e~camination of one of the lesions. At start of the chemotherapy
(bleomycin, vincristine and adxianaycin8 intravenously) ~S had progressed to
about 1a4 cutaneous lesions. The interval between the courses of
chemotherapy was three weeks and stopped after the fifth course. Several
2o lesions had disappeared by three weeks of therapy and complete remission
was
gradually reached after one year.
During chemotherapy several biopsies were taken. The first biopsy was
obtained 24 hours after start chemotherapy (named KS1) and the second
biopsy after 48 hours (named KS2). All biopsies were #lash-frozen in liquid
nitrogen immediately after surgical removal and stored at -80°C.
Diagnosis of
Kaposi's Sarcoma was con~.rmed histopa.thologically.
i
CA 02369183 2002-O1-23
1
Control SAGE libraries IiS3 and KS4 were made from frozen material taken
at autopsy from two AIDS patients with Kaposi's Sarcoma, both of which died
in 198$ without having had anST form of chemotherapy or retroviral treatment.
Example 2
The expression profiles of the biopsy samples were determined using the
SAGE technology. All biopsies were cut with a microtome in 15-~0 lCm sections
and transferred to a tube cox~taining TRIzoI. hh,TA isolation with Tl3,Tzo1
was
performed according to the manufacturer's instructions. Poly (A) RNA ~sxras
obtained using the Micro-1~'ast'I'~rackT~i 2.0 naRNA Isolation Kit. cDNA
preparatiox~ and the subsequent steps were performed as described by
Velculescu. Prixuary analysis of the sequence results was performed using
software especially designed for SAGE by the Bioinformatics I,abaratory of the
academic Medical Centre, Amsterdam (vari Kampen et al, USAGE: a web-
~5 based approach towards the analysis of SAGE data. Bioix~formatics, in
press).
The libraries were also analysed using the Human Transcriptom.e Map {HTM),
a program de~elaped in the Al.'IrIC, which maps TAG's onto human
chroznoaomes (Caron et al. The Human Transcripto~ne lVlap reveals a
clustering of highly expressed genes in chromosomal domains. Submitted for
publication).
We sequenced ~ 47,600 TAG's from the four biopsies, 4'7,268 TAG's &oxu FiS 1
library, 48,671 front the KS 2 library, 49,333 TAG'e from the KS8 library, and
48,814 TAG's from the KS4 library. TAG lists (i:e, individual TAG's plus the
2~ nuxxtber of appearance) were compared with each other in ~JSAGE, TAG
sequences with the highest counts were identified with the amet2g database
available in USAGE (~rhich is an improved TAG identification compared with
the S AGExn.ap database from CGAp (available fram Genl3ank). Secondly, TAG
lists were mapped to chromosome locations with the HTM program, and at the
V
CA 02369183 2002-O1-23
same tizue compared with specific TAG lists (e.g. vascular endathelium,
publicly ava.ilable), and with a compilation o;E' all TAG lists in the
SAGEm.ap
database (designated "All" in HTIV.~ TAG's belonging to genes specifically up
regulated in KS3 and KS 4 identified (Table I): Nucleotide 15 was detexmined
from the original diTAG list in USAGE. The TAG sequence of 15 x~x. was
cheeped with GenBanl~ ($r.AST) to confirm its identification. _4 few TAG's
wexe elixuiuated because of ambiguity in the 15~ nucleotide, or because of
misidentification.
Ex$mple 3
Result o~ the analysis showing the identifiable TAG's derived fxom l~nown
genes with increased, expression in Kaposi's Sarcoma SAGE libraries KS3 and
KS4 compared to libraries KS1. and KS2. The TAG numbers ars first
x~oxtnalized to a level of 100.000 tptal TAG's sequenced pex library to allow
comparison between the libraries and comparison with other libraries in the
public domain.
The sequence catg precedes each TAG sequence given in column 2 of table 1.
CA 02369183 2002-O1-23
1~
Table 1. Overview of identifiable TAG's over-expressed in SAGL~ libraries K53
and KS4.
No. TAG sequenceUnigene ID overe~cpressiou
(5' -~ 3'? xr:o. factors
1. ccccagtcggcHs17Ia96 Ep~ 3
2. citgacataccHs171895 Dual speciiiciry pl'osphatase3
3. catcacggateHs8?112 IIJI receptor. type 10-30
12
4. ggccaaaggccHs'18436 ~ph81 >2
5, ttgcatatcagHs82237 AT group D protein 10 - 15
6. ccctgttcageHs788?4 Tie 12 2-5
7. gatcaatcagtHs16530 Small ind
. cytokiue A18 10-20
8. gagggtgcca&Hs898 Compleiuentcomp. iQj3 5_10
9. taaacctgetgHs99923 galectin 7 g-IO
10. gtggccagaggHs~.420 - FGFT~.3 2-5
11. tctggcccagcHe183 DA~G (DAY blood gxoup) g-10
12. caggtcgctacHs75Ofi6 'frans~n 2-6
13. gageagcgcccHs1I2408 Psoriasis (5100 A7) ~ 20 (speci.&c)
14. acttatt$tgcHs7f152 Decoxin 2-10
1~_ caggcetggccHa74649 CYtochrn~ne C oxydase 2-4
subunit VIe
16. gtgcggaggacHs1810f2 Serum amylaid A1 5-14
I7. acageggcaatHs74316 Desmoplak:n g-L0
18. gatgtgcacgaHs 117T>9 ~er$tin 14 10-14
19. oaggtttcataHs24S95 Small ind. cytnkine, 5-10
814 (BR.A~
20. aactctgaeecHs93675 hecidual proteiz~ induced3-10
by
pmgesterpue~
1. 7AG nurabers of appearance were normalized to iibrary sizes of 100.000
TAG's.
2. Identified as Pan ISldothelial Markers bar St. Croix et al., Genes
expressed in human tumor andot6etium.
b Science 289:1 s 97-1242, 2000.
D c
CA 02369183 2002-O1-23
Example 4
Result of the analysis showing the non-identifiable TAG's derived from EST's
of genes with zxnknown function with increased e:~pression in Kaposi's
5 Sarcoma SAGE libraries KS8 and KS4 compared to libraries KS1 and X52.
The TAG numbers are first normalized to a level of 100.000 total TAG's
sequenced per library to allow comparison between the libraries and
eomparisox~ with other libraries in the public do=nain_
to
The sequence catg precedes each TAG sequence given in column 2 of table 2.
Table 2_ Clverview of identifiable TAG's over-expressed in SAGE libraries ~S3
and I~a4.
No. TAG Unigene no. TD uverexpressian
sequex4ce faatoxl
5~ -.~ 3~)
1. aaatcaataca ~s949o3 EST 4-10
2. tggtaactggc Hs108?41 ESfi 4-10
3. tctgcactgag Hs173?89 ESA' 2-4
4. caggctgctgg T3s60440 EST 4-30
5. atgacagatgg 1-Tsl3??5 EST 5-10
6. gcacaacav.agaHs236510 EST 3-10
7. caaca.ggagaaHs235?9 ES'f 4-10
8. ctgtgcggaae I~s46987 EST 2-10
9. gatggctgect T~s1.8104 ~s~r 4_20
_
10. ctccattgcca Hs31869 ES'r 2-10
11 acctccactgg Hs i 1?457 EST Unique2
1. TAG numbers of appearance were normalized to library sizes of 100.~C10
TAt,~'s
2. This TAG does not appear in any other SAGrE library than. our own.
15 libraries and seems to be a unique new indicator gene fox azxgiogenesis.
3
CA 02369183 2002-O1-23
Example a
liaposi's sarcoma shin tissue was obtained from the same two AIT)S patients
mentioned in example l from whom SAGE ~.ibraries K~S3 and Ii.S4 were made.
Both patients were homosexual men and w ere infected at the beginning of the
HTV-I epidemic in Europe. Patient 1 born in Indonesia was demonstrated to be
HIV-1 positive in 1982. In February 19$5 a histological examination confirmed
the diagnosis of Kaposi's sascpma. He died 13 month latex and postmortem
e~tamination revealed morphological variants of visceral liS. Patient 2
presented in February 1984 at the Academic: Medical Centre with progressive
li.S shin lesions. Dozing follow-up the KS progressed to the intestines,
oropharynx, lung, tongue, sinus piriforxnis and lymph nodes. In Il~Tarch 1586
the patient died. arid autopsy took place. The biopsies of said two patients
were
named KS3 and KS4.
Nor~xal adult breast skin tissue was obtained as discarded tissue from
reduction mammoplasties (obtained from the department of plastic Surgery of
our hospital). RNA isolated of three breast reductions cv~as used to construct
the normal skin expression profile iibra~.
~0
The e~pressian profiles of the biopsy samples were determined using the
SAGE. technology as described in eRample 2.
We sequenced ~ 47,000 TAG's froze the four biopsies, 49,335 TAG's from the
KS3 library, and 48,814 TAG's from the ~.S4 l.ilarary. TAG hists {i.e.
individual
TAG's plus the number of appearance) were compared with each other in
TJSAG~, TAG sequences with the highest counts v~e.re identified vvitb the
axnct2g database a~railable in USAGE (which is an impro~red TAG
identification compared with the SAGrI ;map database from CLAP (available
from GenBazkk). Secox~.dly, TAG lists were mapped to chrouxosame locations
with the H'T'l~i program, and at the same time compared with specif c TAG
f a
CA 02369183 2002-O1-23
22
lists (e.g. vascular endothelium, publicly available), and with a compilation
of
all ~'AC'x lists in the SAG>fmag database (designated "All" in I~TM) TAG's
belonging to genes specifically up regulated in liS3 and KS 4 identified
(Table
1). l~'ucleocide 15 ~aras determined from the original diTAG list in xJSAGE.
The
TAG sequence of 15 nt. eras checl~ed with ('xenBaxxk ($LAST) to contxrm its
identification. A fevsr 'T'AG'S ~cvere eliminated because of ambiguity in the
15~h
nucleotide, or because of misider~tificataon.
example 8
Result of the analysis showing the identifiable TAG's derived from known
genes with increased expression in Kaposi's Sarcoma SAGE libraries KS3 and
KS4 compared to the public libraries of Naiional Center for Biotechnology
Ix~formatzon, 1'he TAG numbers are first normalized to a level of 1~Q.000
total
fiAG's sequenced per library to allow comparison between the libraries and
1~ comparison with other libraries in the public domain.
The sequence catg precedes each TAG sequence given in column 2 of table 3.
Y
CA 02369183 2002-O1-23
23
Table 3. Overview of identifiable tag's overexpressed in SAGE libraries IiS3
and KS4
Tag ~'ag sequenceUnigene IA overexpressed
number no. I
TAGDO gatgtgcacgaHSI17729 ~ieratin I4 10-14
r
TAGO10 cceeagtcggcHs17I596 E h A2 (an 'o enesis) 3
TAGO11 cttgacataccHs171695 Dual specificity phosphatase3
TAGO1'2 catcacggatcgSg2I12 ILI rece tar, ' a 1* 10-30
TAG013 g9'ccaaaggccHs7E436 Sorting nexin 17 >2
TAG014 ttgcatatcagHs82237 AT rou Ia rotein 10- 15
TAG01,5 'ccctgttcagcH$7gg24. Tie I*(angio~enesis)
TAG016 gatcaatcagtH5,16~gp Small ind. cytokine 10-20
Al$
TAG017 gagggtgccaa$s898 Complement COmp, 1t1I35-x0
TAG018 taaacctgctgHs99923 galeatin 7(s ecifia) 8-IO
TAG019 9tggcca~3aggHsI420 FGFR3 (activated in
carcinomas, angiogenesis}
TAGQ20 ~CtggCCG3gCHslg3 D.A~.C (Daffy blood
group} 8-10
TAG021 caggtcgctacHs75066 Translin(invowed in 2-6
translocations
TAC'x0229agGagcgccc~s1,12408 Psoriasin (5100 A7} ~ 20 (s ecific}
TAG033 acttattatgcHs76152 Deeorin (connective 2-10
tissue}
TAG034 caggcctggccHs288761 Hypothetical protein 2-4
FL~'21749
TAG085 gt~cggaggacHs181062 Serum amyloid AI - 5.14
-
TAG03B acagcggcaatHs7g316 Desmo la.kin 5-10
TAG037 caggtttcata~Is24395 Small ind. c~tokine, ~-10
B14
B ~.K
TAG088 aactctgacce~s93673 Decidual protein induced3-10
by
progesterone's
~'rdenti~.ed as Pan Endothelial Markers by St. Groz~ et al., Genes aapressed
in
human tumor endothelium. Science 2$9:1197-1202, 2004.
CA 02369183 2002-O1-23
24
Example 7
Result of the analysis showing the non-identifiable TAG's derived from EST's
of genes with unknown function with increased expression in h'aposi's
Sarcoma SAGE libraries KS3 and KS4 compared to librax-ies ~S1 and KS2.
The TAC,r numbers are first normalized to a level of 100.000 total TAG's
l0
la
2o
sequenced per library to allow comparison between the libraries and
comparison with other libraries in the public domain.
The sequence catg precedes each TAG sequexice given in coluuzn 2 of table 4.
Table 4.
Overview of idenc.ihable tag's over-expressed in SAGE libraries 153 and 11S4
Tag Tag aeguenceUnigene ~ overexpxessed
number. (~'~3') no. ID
TAG023 aaatcaataca $rsgg953 EST 4-10
TAGO'24 tggtaactggc Hs108?41 EST' 4-10
TAG0~5 tctgcactgag Hs113789 EST Z-~
TAG02fi caggctgctgg Hs60440 EST 4-30
TAG027 atgacagatgg Hsl3??5 EST ~-10
TAG028 gcacaacaaga Hs236510 EST ~-IO
TAGO?g ccacaggagaa HS~357g EST 4-10
TAG030 ctgtgcggaac Hs46987 ES'Y'2-10
TAG031 gatggctgcct Hs18104 EST' 4-20
'rAG03? ctccattgcca 11s31889 EST 2-10
TAG004 acctccaGtgg Hsll?45? EST' ITni ue*
*This tag does not appear in any other SAGE library than our own libraries and
seems
to be unique new indicator gene for angiogenesis,.
The overexpressed TAG's listed in tables 3 arzd 4 are the same as in tables 1
azxd 2, respectively. This showtrs that the expression. pattern after
treatment is
CA 02369183 2002-O1-23
comparable with the expression pattern of healthy individuals with normal
expression patterns.
5 Example 8
Using an AT-PCR based method we were able to determine that TAG 11 (table
2) / TAG 004 (table 4} indeed repxesents a differently expressed gene, RNA was
isolated from a KS lesion and the first strand c~h1'A synthesis was primed
with
an oligo(dT) primer with a 5' M13 tail (5'CTA CxTT GTA AAA C .GA CGrG CCA
IO G-(T)$4 8'). Ten microliter total R,1~IA was used, plus primer and 5 ~.1 RT-
mi~c (50
mM Tris, pH 8.3, 75m?VI KCl, 3 m~.'V! MgCI~, 10 mi~2 T.1TT), 80 mll~I dlVTPs
and
24 units RNAsin were added, followed by au incubation for 3 minutes at
65°C
and chilled on ice. The RT reaction starts by adding 5 units AMV RT followed
by an incubation of 45 minutes at 42°C. For the PCR we used a 19-base
TACr-
la specific primer (which consisted of 11 nt identified in the sage with a 5'
l~'L~LII
restriction site and 5 inosine nucleotides to increase the annealing
temperature of the primers) and the -21M13 primer. The RT-mix was added to
80 E.il PCR mixture containing the I00 ng of each primers (-21.m1.3 pRIiI~IER:
5'
GTA AA.A CGA CGG CCA GT 3' and 5' IIT IIG' A'1'G ACC TCC ACT GG 3'),
2a 80mM Tris (pH8.3}, 20 m.'=Vt KCl, fl.l mg 8SA per m3, dN''I'p's (0.1 mM
each), 2,4
~1 ~IgCla, and 2 units Taq polymerase. After incubation of 5 minutes at
94°C,
the reaction was subjected to 35 cycles of amplih.catiou. in a thermocycler
(9700
Perkin-Elxnex). A cycle included deuaturation far I minute at 95°C,
annealing
for 1 minute at 55°C and extension for 2 minutes at 72°C. The
last cycle was
~5 followed by ?2°C incubation for 10 minutes.
The ampli~xed fragment oiras cloned in to an AT plasx~iid (In~i.trot,~eri) and
subsequently the insert was sequenced using the dye terminator sequencing
kit from Applied 8iosystexns Inc, The fragment appeared to have a lextgth of
P a
CA 02369183 2002-O1-23
2s
102 base pairs and the sequence analysis of the fragment revealed the
sequence as depicted in figure 1. This sequencE; rwas identical to axe SST
sequence identified from h'urnan foetal heart (GenBank acc: # AI2175fi5 and
others), which in turn matched a predicted exon on chromosome 19. A relation
with angiagenesis has not been described previously and is new.
Example 9
Confirmation of the identit~r of tag sequences.
A sequence consisting of 15 nucleotides should be enough to identify a
to particular specifzc xnRNA or gene. To confirm the identity of the tags we
developed a RT-PC13. using an oligo24dT pxi~uer for the ~t.T-reaction. ~
5'primer
containing the tag sequence itself is used for second strand synthesis. The
oligo24dT primer is extended at the 5'site extended with a -27.M13 sequence.
In the PCR following the RT-reaction -21M13 primer is used for the
amplification together ~cxrith the 5'primer containing the tag sequence. The
5'primer contain.zng the tag sequence is e~tex~ded with 5 Inosines at its
5'site to
enlarge the binding capacity of this primer. The sequence of the amplified
fragment can be determined to cod. that this is the gene as identified by
the tag sequence. The RT-PCR reactions to con:~rm the tags were performed on
?0 the same tissue samples that were used to prepare the expression profiles
of
tag sequences.
Procedure:
Common buffers used throughout the experimex~ta:
2~
lOx R.T huffex:
5~9 ra~M TRIS, pI-I 8.3
754 mlVl ~Gl~
- 34 xnM MgCl2
a
CA 023691832002-O1-23
- 100 mhI DTT
10x 1'CR buffer:
- 200 mNI TRIS p~i 8.3
s - 500 rnll~x I~Ch
- 1 mg/azl BSA
1 ml TRIzoI reagent (Invitrogen Life Technologies, cat. no. 15596) is added to
10-100 mg tissue or approximately 107 cells immediately (tissue is sliced
141xm
thick by xnicrotome).
The Total RNA isolation of the samples is performed according to the
manufacturers protocol as follows:
~ Add 0.2 ml of Chloroform (Il~lerck) and shake the tube vigorously by
hand fox I5 seconds.
IncubatB for 5 minutes at 1'~T.
Centrifuge the sample at no more than 12,000 x g for 15 minutes at
4°C.
~ Transfer 600u.1 of the colourless upper aqueous layer to a new tube. The
lower organic layer should be red.
~ Ad 0.5 ml isopropyl alcohol (Merck) and mix.
Incubate at xooxn temperature fox 10 minutes.
~ Centrifuge at no more than 12,000 x g far 1.5 minutes at 4°C.
~ Discard the supexxiatant and wash the RNA pellet with 1 ml 80%
ethanol by vortexing and centrifuge at no more than 7, 500 x g at 4°C
for
5 minutes and discard the supernatant.
~ Place the tube at 56°C for 3 minutes to dry the pellet and proceed
with
the Poly A,+ Ii.NA isolation as described i~ the next section.
CA 02369183 2002-O1-23
28
The Poly A~ mRNA isolation was performed according to the manual of Micro
Fasttrack ~ 2.0 Poly A+ mRNA 2.0 isolation kit; as provided by the
manufacturer (Invitrogen Corporation, Carlsbad USA; cat no Fi1520).
Subsequently the isolated poly A+ RNA was used as input for analysis in a R'T
PCR reaction. The RT-PCR reactions started uTith the follovsring mixture of
ingredients:
21M13POLYT primer (140 ngl~z.l) 1.25 p1
x RT buffer 2.0 p1
100 mM dNTP (Pharmacia) 0.8 izl
U RNAsirx (R,oche) 0.3 u1
dH:O (Baler) 0.65 p1.
10 ~ Add 10 u1 of Poly A+ mPvNA dilution to 5 ~l of RT-zni~s.
~ To anneal the primer to the template incubate the reaction mixture at
65°C for ~ minutes followed b~ cooling down to room temperature.
Add 5~t.1 1LTI1.~1 AMV'-R'T' to the reaction znixtnre and perform the Reverse
Transcription by iueubattng at 42°C for 4~ minutes.
1.5 . After the Reverse Transcription ixamediately incubate the mixture at
95°C for 5 minutes to stop the reaction. Ther1 let the reaction cool
down
to room temperature.
~ Add 80 ~J. of PCR-mix to each reaction mixture (total volume is 100 p.1.):
Prepare the PCR-mix per reaction as follows:
- 5' prixnex (100 nglpl), see table 5 1,0 lxl
- 211~I7.3 (1.00 ng/pl.) 1.0 w1
- lOx PCR buffer 8.0 ~xl
- 100 m'!VI MgCh ~. ~. ~.1
- Axnplitaq 5U1~.~.1. (Parkin Elmer) 0.4 u1
- d.~a0 (Baker) 67.5w1.
f ,
CA 02369183 2002-O1-23
29
~ PCR, amplification was performed in a 9700 DNA thermal cycler (~'erkin
Elmer) according to the following program
minutes 95 °C
- 1 minute 95 °C; 1 minute ~5 °C; 2 minutes 72 °C, fox 35
cycles
5 - 10 minutes 72 °C
The TA-cloning of the RT-PCR products was performed according to the
manual of TUl'O TA Cloi»g~ kit as provided by the ananufactuxex (rnvitrogen
Corporation, Carlsbad 'USA; cat no K4600) using 'the pCR,~ IT-T4P0~ Dual
promoter sector and the TOP10 One Shots Cells:
Screening of the clones was performed using ~'CFt with SP6 and T7 primers.
As follows:
~ ltesuspend the colony in 50p.1 dH20 (Baker}. Add 1 E~l of this bacteria
suspension to 10 F.il of PCR mixture.
. Prepare the PCR-mixture per reaction ~~s follows:
sPS (loo ngyl) o. la
p1
T7 ( 100 r~glpl} 0.10
~.1
lOx PCR buffer 1.00
p1
100 ~zvi ~ugclr o.20
~.l
100 mM ch~l'~'1''s (1'harmacia)0.08
~.M
Amplitaq SLTlp.1 (Perkin0.04
Elmer) ~zl
dHaO (Baker) 7.48
u.1
~ ~'CR amplification was performed ixi a 970 DNA thermal cycler (Perkin
Elmer) according to the following program.
- 5 minutes 95 °C
- 30 seconds 95 °C; 80 seconds 55 °C; I minutes 72 °C,
for 25 cycles
- 10 minutes 72 °C
~ Run 5 p1 of the Colony-PCR product on a. 1.6% a.garose 1~TBE gel
stained with EthidiumBromide.
! A
CA 02369183 2002-O1-23
so
~ '4 isualise the amplification products on a UV-illuminator tv identify
insert-containing clones.
Clones containing ixuert were sequenced fmm both directions using Sl'6 and
T7 primers and the r'~BI Prism $ig-Dye terminator cycle sequerxciug kit
(Pexl~in Elmer Applied Biosystems, Faster City, Calif T~T~A).
CA 02369183 2002-O1-23
31
Table ~: Primers used ~or tag confirmation
Ta namz Seguence ID
-?1M13POLYCTA RT-primer
GTT
GTA
AAA
CGA
CGG
CCA
GTT
TTT
TTT
TTT
TTT
TTT
TTT
TTT
T
AG007 III ratin 14
IIC
ATG
GAT
GTG
CAC
G
AG014 III a brie A2 am. 'o enesis)
TIC
ATG
CCC
C
AG
TCG
GC
TAGOll II dual. s e~ei hos hatase
IIC
ATG
CTT
GAC
ATA
CC
TAG022 IIIIIC IL1 rece tor, a 1.
ATG
C
AT
CAC
GGA
TC
_ IIIIIC Sortie Neon 19 (SNX17)
TAGO13 ,ATG
GGC
c~A
AGG
CC
AG814 III T ou D rotein
IIC
ATG
TTG
CAT
ATC
AG
AG015 III 'e 1 (an 'o enesis)
IIC
ATG
CCC
TGT
TCA
CxC
AG016 III small ind. C tokir~e
IIC A18
ATG
GAT
CAØ
TCA
GT
AGOT7 III om Iement com . 1 beta
IIC
ATG
GAG
GGT
GCC
A_~
TAGO18 III alactin 7 1i et to
xTC
ATCx
TAA
ACG
TGC
TG
TACx019 III FGFR3 11 et to )
IiC
ATG
GTG
GCC
AGA
GG
TAG020 III DARC
IIC
ATG
TCT
GGC
CCA
GC
TAG021 III TTanslin
IIC
ATG
CAG
GTC
GCT
AC
T:~GO.''.2II Psoriasiiz (5100 A7)
IIC f 1l et to
ATCx
GtIG
CAG
CGC
CC
TAG033 a Decorin
~ IIC
ATG
ACT
TAT
TAT
GC
TAGr03.~ II H othetital rotein FLJ21749
iIC
ATG
CAG
GCC
TGG
CC
TAG035 II G CxTCx CGrG AGGAC Serum ~m loid
IIC
AT
TAG036 II 'G ACA GCG GCA AT Desmo~Tal~
ITC
A"!
TAG037 II Sxaall irsd. C kzne.
IIC B14 RAKE
ATG
CAG
GTT
TCA
TA
TAG038 a ecidua.l protein induced
IIC by
T:~G023' ATG ro esterone
AAC ST Uni ene zxo_ Ha94953
fiCT
GAC
CC
II
IIC
ATG
AAA
TCA
ATA
CA
AGfl24 II ST U ' ene no. Hs108741
IIC
ATG
TGG
TA.A
CTG
GC
AG025 III S~' 'Uni ene zoo. Hs173789
IIC
ATG
TCT
GCA
CTG
AG
AG026 III EST Uni ena no. Hs60440
13C
ATG
CAG
GCT
GCT
GG
AG0~7 III EST Uni ne no. HsI3776
IIC
ATG
ATG
ACA
GAT
GG
TAG028 III EST Ur~.i ene no. Hs2S6510
IIC
ATG
GCA
CAA
CAA
GA
TAG029 II EST Uni eue no. Hs23579
IIC IG
ATG
CCA
CAG
GAG
AA
TAG080: II EST U ' ne no, H's46987
IIC
ATG
CTG
TGC
GGA
AC
TAG031 II EST D'ni ene no. I~s18I04
IIC
ATG
GAT
GGC
TCrC
CT
TAG032 II Ha3186$ s' lec-1 ar
IIC sialosdheai.n
ATG
CTC
CAT
TGC
CA
TAG004 II EST Uni exfe no, Ha1124557
IIC
ATG
ACC
TCC
ACT
GG
.
CA 02369183 2002-O1-23
32
Results of Confirmation of tae sequences:
Of 18 ag sequences that were analysed with the protocol as described in this
example 14 were confirmed with sequences analysis using the R.'1'-PCR with
the oligo24dT primer and t3~e tag-based primer as described above (tags
designated 004, 007, 011, 012, 014, 015, 016, 017, 022, 025, 029, 030, 032,
036
in table 5). Four tag sequences could not be confirmed based on this method
(designated 010, 013, 018, 019). This was probably due to the fact that the
tag-
based primer was not specific enough or that the polyA tail of the mR~lA was
not long enough. Fox one tag an alternative more specific 5'primer was
to designed to perform a RT-PCR together with -21Nl13POLYT (tag designated
004). For tag010 a complete specific primer set was designed to perform as
well the Reverse Transcription as the amplification. Other tags were
confirmed by using a specific RT-PCR. primer set followed by a Nested PCR
tvith a speci_~c nested primer set (tags designated 013, O1$, 013). Sequence
results of all confirmations are listed below a:od in the figures. The tag
sequex~ce in the mRl'~IA sequence if present is shown in bald fonts.
TAG004 (EST AI21?565, genbarak number BF468?28):
Confirmed with protocol from this example.
CATG~.GCTGCACTGGA.AGAGGGGGCTAGCGTGAGCGGTGATTCTC.A..ACCT
ACCATAACTC'I"FTCCTGCC'1~CAGGAACTCC A...aT_4AA4CAT'I'~I"~CC A.TC CA
(Figure 1)
TAG00? (keratin 14, genbank number ~M_008a78):
Confirmed with protocol from this example.
CATGGATGTGCACGATGGCAAGGTGGTG'T'CCACCCACGAGCAGGTCCTT
CGCACCAAGA.4CT'GAGGCTGCCCAGCCCCGCTCAGGCCTAGGAGGCCCC
CCGTG'x'C'xGACACACrATCCCACTGrGA.AGATCCCCTCTCCTGCCC_AAGCACT
TCACAGCTGGACCCTGCTTCACCCTCACCCCCTCCTGGCA.A.TCAATACAG
34 CTTCATTAr!'C'1'CxAGTTCxCTAAAAA_A.AAA..A..AAAA_
(Figure 2)
,
CA 02369183 2002-O1-23
33
TAGO10 (ephrin A2, genbank number ~I Oa2088):
The RT-PCR with the 5'prixner designed on the catg-site (the original primer
shown in table 5} gave no confirmation. Most probably the tag-based primer is
not specific enough. Ephrin A2 specific primers were used for confirmation.
The tag sequence is clot included in the Ephrin specific RT-PCR.
ATCTACCAGCTCATGATGCAGTGCTGGCA.GCAGGAGCGTGCCCACCGCCC
CRAG'r'TCGCTGACATCGTCAGCATCCTGGACAAGCTCATTCGTGCCCCTG
ACTCCCTCAAGACCCTGGCTGAC'T"t"1'GA.CCCCCGCGTCxTCTATCCGGCTC
CCCAGCACGAGCGGCTCGGAGGGGGTGCCCTTCCGCACGGTGTCCGAGT
GGCTGGAGTCCATCAAGATGCAGCAGTATAGGGAGCACTTC
(Figure 3)
TAGOI.l (dual specificity phosphatase, genbaxik number ~h0a3720):
16 Confixmed with pxotocol from this exana.ple.
CATG C '~TGACA'~A C CTAC C AGT ATTATT C C C GACG AC A CATATA CaTATG
AG_4ATATACCTTATTTATTTTTGTGTAGGTGTCTGCCTTCACA_A.ATGTCAT
TGTCTACTCCTAGAAGAACC_4A.4T AC CTCAATTTTTGTTTT'1'G AGT ACTGT
ACTATCCTGTAAATATATCTTAAGCAGG'1"f"~'GTTTTCAGCACTGATGGA..~A
2o ATACCAG'Z'GTTGGGTTTTTTTTTAGTTGCCA.<1CAC'rtTCxTATGTTTGCTGAT
TATTTATGACCTGA..g.ATAATA'T'ATTTCTTC'1'TCTAAGAAGACAT'I'~"I'GTTAC
ATA.A.GGA'FGACTTTTTTATAC_4ATGGAATAA.ATTATGGCATT'~CTATTG
(Figure ~)
25 '~'AG012 {~~1. receptor, type 1, genbank numbEr X1~I_U02686):
Confirmed with protocol from this example.
CAS'GCATCACGGA'I'CAATAGACTGTACTTATTTTCCA_4TAAA.9.TTTTC_4A
ACTTTGTACTGTT
(Fz.guxe ~)
so
a a
CA 02369183 2002-O1-23
34
TAGOI3 (ephrin B1, genbank nuxubers XM_002o35, BC002524):
The RT-PCR with the 5'primer designed on the catg-site (the original primer
shown in table 5) gave no confirnxatian. Most larobably the tag-based primer
is
not speei$.c enough. Ephrin BI specific primers Were used for conf.rmation:
The tag sequence is included ix~ the 8'primer of the Eghrin B1 speeif c RT-PCR
fragme~xt. The Nested PGR. fxagment is shown here and is just located
upstream of the tag sequence.
AACTTGCCCTGTGCCTGTGTCCCCCATG'rCTACxGGGCGGAGGGGTCTTTTC
CTTCTTCTTTCC'1"ACCTACCCCfiTTTCTCTTGGCCAGGCxG CGTCGTATGCT
~o ACCTTTCCTTGTCCCCTGGGCTGGCTGCACAGAGGATTGCCCCTTCTCTTT
TCAG AG CTGGCCCTCGATGCCAAATTAGCATTTAtarTA,TTTTGCTCAAACxTC
TAAGGGACC
(Figure 6)
15 TAG014 (AT groug lJ protein, genbanh numbers XM_406184, AF230388):
Confixmed with protocol fram this example.
CATG'~"~'GCATATCAGGGTGCTCAAGGA'T'TGGAGAGGAGACA.AA_4CCAGG
AGCAGCACAGTGGrGGACA'T'CTCCCCxTCTCAACA.GCCCCAGGCC'~'ATGGGG
GCTCTGGA.4GGATGGGCCAGCTTGCAGGGGTTGGGGAGGGAGACATCCA
20 GCTTGGGCTTTCCGCTTTGGA.ATAAACGATTGGTCTG'rCACAAA_AAAAA.AA
('Figure '1)
TAGOx5 (TAE 1, genbank number XM_002057):
25 Confirmed with pratocoi from this example.
CATGCCCTGTTCAGCTACTCCCACTCCCGGCCTGTCATTCAG.A..4A.A,AAAT
A-~1A'1''GTTCTAATAACxCTCCAAAAAAAA.9AA.t~AAA_9AAAA_~AAA
(Figure 8)
CA 02369183 2002-O1-23
~5
TAG016 (small ind. Cytol~ne A18, genbank numbers ~i_008451, Y13 r 10,
AF111198}:
Confxrm.ed with protocol from this example.
CATGGATCAATCAGTGTGATTAGCT'TTC'I'C AGCAG ACATTGTGCC ATATG
T ATCAAATGACAA..~TCTTT ATTG.AATGGTT'T'TGCTC~.GCAC C:4C CAA
TATATTGGCAGTACTTATTATATAAAAGGT..:~A.ACC AGCATTCTCA_4.~AA..~~1
AAA.A.4AA
(Figure 9}
TAG017 (ct~mplement camp. 1Q beta, genbank nuxn.ber X VI_010E60):
Confirmed with protocol from this e~auaple.
CATGGAGGG'r'GCCAACAGCATCTTTTCCCiGrCxTTCCTGCTCTTTCCAGA~'
ATGGAGGCCTGACCTGTGGGCTGCTTCACATCCACCCCGGCTCCCCCTGC
CAGCA.4CGCTCACTCTACCCCCA.ACACCACCCCTTGCCCAGCCAATGCAC
1~ ACAGTAGGGCTTGGTGAATGCTGCTGAGTGAATG~ACxTA.~-LTA.~ACTCT'1'C
A.4GGCC
(Figure 10)
TAG018 {galectin 7, genbank cumbers 1~-00:280?, 'U'OS643):
The RT-PCR ~rith the 5'primer designed on the catg-site (the original primer
shown in table 5} gave no confirmation. iVlost probably the tag-based primer
zs
not specific enough. Galectin 7 specific primers were used for confirmation.
The 5'primer used in the Galectin 7 specific RT-PCR eantaius Che tag
sequence. The tag sequence is included in the 5'primer of the Galectixt 7
speci$c RT-PCR. fragment. The Nested PCR fragment is shown here and is just
located downstream. of the tag sequence.
CGGCTGGACACG'x'CGG.AGGTGGTCT'TCAACAGCAAGGAGCAAGGC'T'CCT
GGGGCCGCGAGGAGCGCGGGCCGGGCGTTCCTTTCCAGCGCGGGCAGCC
CTTCGAGGTGCTCATCATCGCGTCAGACGACGGCTTCAAGGCCGTGGTTG
CA 02369183 2002-O1-23
36
GGG ACGCCCAGTACC ACC ACTTCCGCC
(Figure 11)
TAG019 (.~'Gp'R.3, genbank numbers NM 02965, ?vFM 00012):
The RT-PCR with the 5'primsr designed on the catg-site (the original primer
shaven in table ~) gave no confirmation. Most probably the tag-based primer is
nat specific enough. FGFR3 specific primers nvere used for confirmation. The
tag sequence is nat included in the Ephrin specific RT-l.'Cl~,. The Nested PCR
frag~zent is shown here and is located upstream of the tag sequence.
~.o CAC.4ACCTCGACTACTACAAGA.4GACAACCAACGGCCGGCTGCCCGTG_.~A
GTGGATGGCGCCTGAGGCATTATTTGACCGAGTCTACACTCACCAGAGTG
AGGTCTGGTCCTTTGGGGTCCTGCTCTGGGAGATCTTCA.CGC'r'GGGGGGC
TCCCCGTACCCCGGCATCCCTGTGGAGG=~GCTCTTCAAGCTGCTGAAGGA
GGGC
(Figure 12)
TAG022 (~'sox~asin (5100 A?) , genbanh. number ~i_048120):
Confirmed with protocol from this example.
CA'X'GGAGCAGCGC CCTGTTC CGGGCxGCAGCCAGTGACC CAGCC CCAC C
AATGGGCCTCCAGAGACCCCAGGA:~CA~~.'_~A'r'GTCTT'CTCCCACC
(figure 13)
TAG025 (EST Unigene no. Hs173789, genbank numbers XM_0~.840~,
AL137262):
Confirmed with pxQtoeol from this example.
CATGTCTGCACTGAGA.~4CTG CATTTCAGTAGC ATTTGTC ATCCAGCCG
GAAGTTAAAGCACACTTACTTTATTCACC'i'AT'1'T'ITATAAT AAACGTTCTT
GCTGCTGTG ~i.~AAAA.A.A~~AAA
(Figure 14)
CA 02369183 2002-O1-23
3'T
TAG029 (PIG, gEnban.l~ cumbers 011453, A.~2~I830}:
Confirm.edvwith protocol from this example.
CATG C CACAGGAGAATTCGGGGATTTGAGTT'I'CTCTGAA'r'AGCATATAT
ATGATGCATCGGAT A.GGTCAT'1'ATGATTTTTTACCATTTCGr.r'1CTTACATr~4
TGAAA:4CCAATTCATTTTA.4ATATCAGATTATTATTTTGTA_AGTTGTGGAA
AA_gG CT A.~TTGTAGTTTTCATTATGAAGT'I~~T CC CAATAAAC C AGGTATTC
TAAACTTGAAAAA.a..c~A.4AA_4AA.A.AI~AAAA_4AA
(Figure 15)
TAG030 (EST 'Unigene no. T~s469$7, genbank numbers DG151190, BG057289,
BE858276, AVBSI759, BE50$169):
Cor~frmed with protocol from this e?cax~aple.
CATGCTGTGCGGAACTGCGTC AGGGC_AAATGTCACAGCAGGATTTCCCC
AACCCAGCTCCATCATCACAGACACAGAGGGCTGCAGGGGAGGCCTGCCC
ACTGTTTTGTCGACTCTGCCCTCCTCTGGCAGCATAGATCCTTAGGTGCTC
AATAA.ACxGTCxTGCTGTAT'TGi~A~~AA.A.k~AAAA..9AAAAr'~.4~1~4
(Figure 16)
TAG032 (SiaIoAdhesin, also called Siglec 1, ger~bank ntux~.ber ~ 016245):
Confirmed with protocol from this example.
CATGCTCCATTGCCAGACTCTTGCTGGGAGCCCGTCCAGAATGTCCTCC
C_4ATAA.A.ACTCCA.TCCTATGACG C~?:A.AAAA_~9.A.~.A.AA.AAAAAA_~,.~..4...A.
(Figure 1'~
TAG036 (Desmoplakin, genbank numbers X11_004463, ~ 004415,
AF189065):
Confirmed with protocol from this exanxple.
CATGACAGCGG CAATCTTTTCTT'r'GCx'z'CAAAGTTTTCTGTTTATTTTGCT
TGTCATATTCG ATGTACTTTAAGGTGTCTTTy4~'GAAGTTTGC2'ATTCTGGC
so AATAAACTTT~'AGACTTTA AAA_.~AAAA.AAAAAAA
).
CA 02369183 2002-O1-23
38
(Figure 1.8)
Example IO
Determination of the gene expression levels of the t~guences in skin
sam s
To get a feeling for the use of the tag sequences as markers for angiogenesis
process skin samples with (o different samples) and without (2 control
samples) Kaposi's Sarcoma lesions were analysed for the expression level of
the genes identified by the tag sequences.
Procedure:
1 ml TRIzoI reagent (Invitrogen Life Technologies, cat. no. 1596} is added lo
10-100 mg tissue or approximately 107 cells immediately (tissue is sliced 14~m
thick by microtome).
The Total RNA isolation of the saarnples is performed according to the
manufacturers protocol as follows:
~ Add 0.2 ml of Chloroform (?Merck) and shake the tube vigorously by
hand for 1~ seconds.
fncubate for ~ minutes at RT.
~ Centrifuge the sample at no more than X2,000 x g for 15 minutes at
4°C.
Transfex 8001 of the colourless upper aqueous layer to a new tube. The
lower organic layer should be red.
~ Ad 0.5 m1 isopropyl alcohol (Merck) and m.ix.
Incubate at room temperature for 10 minutes.
23 . Ce~.trifu.ge at no more than 12,000 x g for 15 minutes at ~°C.
s Discard the supernatant and wash the RNA pellet with 1 ml $0°!0
ethanol by vortexing and centrifuge at no more than 7,500 x g at 4°C
for
~ minutes arid discard the supexnatazxt.
~ Place the tuba at 56°C for 3 minutes to dry the pellet and proceed
wirith
the DNase treatment as described in the next section.
CA 02369183 2002-O1-23
39
To make sure no genomic DNA exists in the Total RNA isolate we pexforra a
DNase treatment. Protection of RNA against RNase activity of DNase 1 is done
by the addition of RNasin to the DNase reaction. The DNase treatment was
performed as follavvs:
~ After TRizol Total RNA isolation resuspend the pellet in $$ E.i.l. d~-I~>O.
Add sequexttially to the R.'~TA solution:
- io ~l lox DNase 'duffer (AAt:abion)
- 1 ~l 40U1u1 R.Nasim {Roche)
to - 1 ua.10 U/~,t DNase r (Rache)
Incubate at 37°C fox 1 hr.
~ Raise sample volume to 200p.1 by adding J.00 ~.Gl dH20.
~ Add 200 p1 cold PhenoUChloxoform pH8 (PC8) and infix thoroughly.
Centrifuge full speed far 6 minutes at rooxa temperature in a
mi.crocentrifuge.
~ Transfer the aqueous top layer to a new microcentrifuge tube add
sequentially:
- 3 u1 glycogen {Roche)
- 100 p,1 10 M ammonium acetate
700 ~.zl 100% ethanol
Mix thoroughly and centrifuge at 25,000 x g far 15 minutes at 4°C
and
decant the supernataxtt.
~ '4'Vash twice by adding 700 u1 80 °/ ethanol. lYiix thoroughly and
centrifuge at 25,000 x g for 5 minutes a.t 4°C and decant the
28 aupernatax~t.
Dry the pellet for 5 minutes at 56°C and resuspez~d in. I1 p1
dI~24.
~ Dilute 1 w1 of the RNA isolate in 140 w1. dHzO and calculate the 12NA
concentration and yield of the isolate through ~I3zso measurement. fibs
yield o.~ DNass treated Total I~NA should at least be 13.5ug for the
30 detexmimation of the complete TAG expression pxofle.
CA 02369183 2002-O1-23
~ Prepare a solution of the RNA isolate with a concentration of 80 ngl~l.
ThlB 13 the starting RNA solution far the series of dilution for the TAG
speci$.c RT-PCR.
3
Per sample of DNase treated Total RNA 1.8 TAG specific RT-PCRINested
pCR reactions are performed in series of dilution of the RNA. For each
sample five contxol RT-PCR's will be performed in series of dilution namely
HIV-1 GAG (SK39/140), t'xAl'DH +RT (in duplo) arid uo-RT reaction (in
duplo). The follovv~ing dilution scheme was used to derived the samples in
serial dilution that are analysed with the RT-PC1't:
1. 270 u1 ~0 ng/~.~1 DNase treated Total RNA DNase treated.
10 ~Cl 50 nglpl solution as input in RT-PCR of each primerset = 500ng.
2. 27 E.~1 50nglul Total RNA 10x diluted = 2'70 girl. 5 ng/~l
3.0 ail 5 ng/j.~. solution as input in RT-PCR of each primerset = 50 n.g.
3. 27 ~.l 5 ng/ul Total RNA 10x diluted = 270 ~.~1 0.5 ngl~l,
la X10.5 ng/pl solution as input in ~tT-PCR of each pri~erset = 5 ng.
4. 27 ~tt 0.5 ug/~1 Total RNA lOx diluted = 2?0 ~,~1. 0.05 ngl~l.
1.a ~.tl Q.05 ngl~.tl solutiox~ as input in RT~PCR of each primerset = 0.5 ng.
5. 2T pil 0.05 nglp,l Total RNA 7.0x diluted = 27a p1 U.00o ng/~.1.
10 ~,l 0.005 ng/~.d solution as input in RT-PCR of each primerset = 0,05
ng.
s. 2~ ~l o.aa5 n~~l Total 12IVA 10~ diluted = 270 E.~,l a.aao5 ngl~l.
2~ 10 f,cl O.OaOa xiglE.~l solution as input in 1~,T-PCR of each priurarset =
0.005
ng.
7. 271 O.OOOa xtglpl Total RNA 10x diluted = 270 l.~l 0.00005 nglpl.
10 ~.el 0.00005 ng/~ solution as input in RT-PCR of each primerset =
x:0005 ng.
$. 27 u1 0.00005 nglp.l Total RNA lOx diluted = 2?0 ~.1 p.000Q05 ngl~,l.
CA 02369183 2002-O1-23
41.
p1 0.00000 ngl~l solution as input in RT-PCR of each primerset =
0,00005 ng.
s TAG specific RT-PCR on Total RNA sexies of dilution using AI~iV-RT was
perfaW ned as follows. The reverse Transcription Reactions of all the T:4Grs
on
DNase treated Total RATA i.n series of dilutiox3 axe performed ix196 wells PCR
plates. Ten ~1 of each Total R.NA dilution is used as input for the RT PCR.
fihe
reaction volume of the Reverse Transcxiption is 20 uI and contains d.NTP's,
r0 ~igCl2 ax~.d RNasixa..
~ Prepare the RT-mix per reaction as follows:
3' primer (Za0 n~'~1) see table 6 1.25 u7
10 x 1'~T buffer 2.0 p,1
lao ra_~rl dNTr' (1'harmacia>0.8 p1
U RN.Asin (Ruche) 0.3 ltl
dH=O (Baker) 0.85
g1
15 ~ Add 10 ~,1 of Total RNA dilution to 5 ~:~1. of RT-mix.
~ To anneal the pxirner to the template incubate the reaction ma.xtuxe at
B5°C fox 5 minutes followed by cool~.g do~rn to room temperature.
~ Add 5.41 lUl~sl AMV-RT to the reaction mixture and perform the Reverse
Txanscripti.an by incubating at 42°C for ~5 minutes.
20 ~ After the Reverse Transcription immediately incubate mixture at
93°C
for 5 minutes to stop the reaction. Then. let the reaction cool down to
roam temperature.
~ Add $0 p1 of PCR-mi~c to each reaction mixture (total volume is 100 u1).
Prepare the f'CR-mix per reaction as folloars:
5' primer (100 ng/gl) aee table 0 1.a u1
lox PCR. buffer s.0 p1
xoo mgt lugch ~..s Wit.
CA 02369183 2002-O1-23
~Z
Amplitaq 5U/~1 0.4 u1
dHaO {Baker) 68. 7 ~
PCfi, amplification is performedin a 9'700 I~N'A thermal cycles (Perkiu
~lmer) according to the folloarin.g program.
a minutes 95 °C
- ~ minute 95 °C; I minute 55 °C; 2 minutes ?2 °C, for 35
cycles
- 10 minutes ?2 °C
Subsequent to this first round of amplification a second rouxtd, nested,
amplification was performed. TAG specific second round nested pCR on RT-
PCR product gas pe.~ormed as fol3.ows:
Add 5 i.,1 of the ~'AGr RT-PCR product io. 9;5 ~.~1. of the Nested-PCR mix.
~ Prepare the Nesied-PCIt rain per reaction as follows:
o' nested primer (100 nglgl),0.5
see table 7 ~l
8' nested primer {loo ngl~zl),0.5
see table 7 u1
10~c PCR buffer 5.0
p7
100 mM MgCL 1.25
l~l
100 mM dNTP (Pharrnacia} 0.4
u?
Amplitaq 5~'lul 0.2
p1
dH20 {BakEr) 37.1a~1
The combination of primers used for amplification of the genes
identified by the tags and the length of the amplified fragment is given
in table 8.
2a
~ PCR amplif eation is peri'or~t.ed i.n a 97x0 I~NA thermal cycles (Perkin.
Elmer) according to the followixtg program.
- 5 xxdnutes 95 °C
- ~. minute 9~ °C; 7. minute 55 °C; 2 minutes 72 °C, for
25 cycles
CA 02369183 2002-O1-23
43
- 10 minutes 72 °C
~ l~,un 10 fcl of the Nested-PCR product on a 1.5% agaxose IxTBE gel
stained with EthidiumBromzde.
Visualization of the TAG amplified fragments in the dilution series on a
~t1'V-illuminator reveals the level of expression by determining the
highest dilution still giving a positive signal.
CA 02369183 2002-O1-23
44
Table fi. RT-PCB?, primer design for &rst roumd amplif~ation.
~,5 lAtoo4G~~lE;;GGC
CTT
TA_A
CAC
CCC
GTT
CCT
_
3'T~1,GOO~GEN~T
CAG
CCrC
TCA~
~'
...
.
,
iTGG
TAG~GTT
GAG
~lA.
.
_
~~._. . _
.
.:~'TAG007GENE-N
'AGGi xlGA
CGA AAG
GTC GCT
ACT GCA
.
3 TAC~r00'GE1VEaCAG
TTC
TTG
GTG
CGA
.EGG
ACC
T
5'TAGOIOGENE..
.
~TC
TAC
CAG
CTC
.4TG
ATC,
CAG
TGC
T
'~
3'T9GOiUGENE_
.~
...
.
~
~GAA
GTG
CTC~~CGTATA
CTG
CTG
CAT
5'TAGO11GENEL4GT
GGG
TAC~ATC
AAG
TCC;
ATC~TG
A
_
3'TAGO3 ~1GENE_
' -
.
,..
-
CAC
~TGCx
TAT
TTT
UCCA
TCA
GTG
.CT
~
-__
...
..
...
...
..
~~
5'TAGQ12GENEh'TAA
AGT
TGT
CCT
GCT
TGA
GCT
GGA
_ _
:3'TAGpI2GENE~~
~
;~GGC
ACG
TGA
GCC
TCT
CTT
TGC
AGT
v5'TAG018GENE-.
.
,.
~CTC
TAC
CCC
AGA
GGA
ATT
TAC
AGA
.
..
i3'TAG013CiElVk,'~GGG
CCA~GAC
CAA
ACA
GACr
ACC
TCT
_
,5'TAGOZ4CrEIrE
'~C'rGC
AAC AAG
CAG AAG
GCG GTC
A
3'TAG0,14GrEN_
E
~
,
;'1'GA
TCT~TGA
GCT~GCA
GCT
GCT
CCT
5'TAG0~5GENECxCT
GGT
CGG
AGA
GAA
,.
i;GAA
TGT
' 3 TAG015GE~TE_,
~ .....~_
~ ~'GG
GGC
AGC
TTT
TCA
TAG
AGC
~'
..
..
..
a'TAG016GE'V'E...
..
~;~~
TCT
GCC
TGC
CCA
CrCA
TCA
TGA
9'TAG016GENE''T'CA
GGC
ATT
~CAG
CTT
CAG
GTC~GCT
~
~4
5 TACr017GENE~G
TC
TCT
ACT
ACT
TCA
CCT
ACC
A
B T~GOZ 7GENE~~._.
,~
_
rTGT
TGG
GGG~TAG
AGT
GAG
CGT
~TGC
T
~o'TAGOisGENE~GCa.
GGT
TCC
ATG
TAa
ACC
TGC
TGT
3'TAG018G,ENE
" '. _ ~jGTG
CTC AGA
AGA TCC
TCA CGG
AGT
~ ' %:
~.5"f ~iG0I9GENE_
. ..
.
_
~GTG
ACC
GAG
GAC
AAC
GTG
ATG
AAG
A
i'3 TAG IiCt~T
ol9GENF GAT
CAT
CxTA
CAG
GTC
~TGr
TGT
_
_....
.
____ ___
i~5'TACr02'~GE~TE
~iTGA GCA
ACA~CTC
AAG CTG
AGA ~G ~~
1- ~ ~~
j
:3'TAG0~2GEh,'~;TCT
CTG
GAG
GCC
CAT
TGG
T
S'T ~1G0'~SGENE~ x9,TG GGCx
; CA GGA ACA ~TCT GGC AGA
T
3'TA(C~2~GrEN'E~' ~GC GGC TGG ATG ACA AAT GCT ACT
~ ~~
5'TAGO_~9G ~ C,rC AGG TTT ATC TGG GCT ~CTA TCA
EIW~ ~u .
_
3'TAGll29GENE. _...~___,__ .~r ....... ..,...-
,;~_.;..:::.~:._::. .::..._:-._.-:._:...~_.:...:._::-..._:,~:::_:.::~.
~ ...... .
T TCA TAA
G?s CCT ATC CGA TGC AT
;!
~
~'TAG030CrE~IE._.., , . ,.,
.~ C'IT GCA
. A.AG ATA GGA GAG GCT CCA
~
. y-_.yATT GAG CAC CTA AG(~ FTC SAT GCT
'~3 T.AGp3aGENE_ ~~~
y
,'I
~5'TAG032G'cEN~~ 't'GC GAA TCA GrGG ACC AAC AGG AGa
>-r - w. .. . ;
k
'e u. ra
ev,
.. .... ~.
. ~ , ,.
. . 4~
;3'TAG032C3ENL,..~:
._.
GQ.A GGA
:CAT
~'~'TG
TCT GGA CGG
GCT ~. '
~
5'TAG036GEN _
.
,
_
;sA'1'T
TAG
CAGr
TAG
TTC
TAT
TC'xQ
GCA
~~
3'TaG036GENE,.FACT
GAT.'1'AG
CAC.TTC
AGA"CCrCyACT_,...
",
..~
~.
~,
~'
CA 02369183 2002-O1-23
40
Table 7. Nested-PCR primer design.
:~5-1'ACxoU~trENE-2SCAT
~ CGA CAA ATT GCG ATC T
.
'3'T~G004GEIV_
t' 2 _ fCGC TAG CCC CCT CTT CC_4 G'X' -
~_~. _. ._. . .. __ ..
~"TAG047GENE 1 AGG AG 4 TGA ~TTG ~GCA GCG T ~ ~
2.
.
__
s3'T4G0U?GENE~GGA GGA.GGT CAC ATC TCT GGA T
2 _
.
~ TAGOlO,C.r~ENE;CCA AGT TCG CTG ACA fiCG T..'. .
~
'a'TAGOioGENE-2LTGC TGG GGA GCC GGA TAG ACA
~
~5'T~GUIIGENE~GAA GAG
2 A.4:A GGA CTC AGT GT _.
_, ,
_ 3
' ~GA TAT ATT TAC AGG ATA GT
'TAGO11GENE
2
_5'~'AG ~tAA1 TCC AAG ACT ATG AGA
t?12GE
_I~TE 2
_ .
_ ;,ACTT AG
. T GGC TGG TGA CAG T
,s'TAGOi2GENE
2
~
:'o'TAGOi8GENE_
2 ':4AC TTG~CCC TGT GCC TGT GT ~ ~ f
v3 TAG013GENE~T CGC ~TTA GAC TTT GAG CA_ _
'~
_
v5'TA.G014GE'~TECTT CTG CGA GCT GCA TCT CA
2 _ Tf
3'TAG014GE~TEC
~ G
'
'
A ~
GT
AC AGC TCC GTC
r
~
'i5 TAGO1~GENEGAGA GGA_ GGT TTA. TGT G_4A GA .
~ "
_
3'TAGalaGET~TE... .. .. . . G
2 aACT ATC TCC .CAA AGA AGG ~,CT
'~5'TAG016GENE~'GT CCT CGT CTG CAC CAT
~ r
3'TAG016GEIVE_ _
2 ' s
1~ATG TAT TTC TGG ACC CAC T ~ ~ ~
~
5 TAG017GE'~TE~
'~ _
~GTC ACC TTG TGT GAC TAT ~GCC T ._
.. _. .. .
'3'TAG01'7GENEACA GGT CAG GCC TCC ATA TCT~
2 ~~
k
_
5'TAG018GEIVE_
2 ~ ~CGG CTG
G AC ACG TCG GA
a3'T~G018G~NEtGGC GGA AGT GCxT GGT ACT _
2
5'TAG019GENL T y
2 ~ j CAC AAC CTC GAC TAC TAC A
3'Tr'~G019GENEj~GrCC CTC CTT CAG CAG CTT~~ " ..
2 .
.. !~i'TC AC,A, AAT ACA CCa GaC~GTG AT
TAr~o~2GENE _.. .
~
s'TAG022GE'~TEGGG CGC
2 TGC TCC ATG GCT~ CTG CT .
,
5'TAG025GENE (iTGC CTA GA.A AGG GGT GGC 'r ;I
2 .
.
3 TAG02~aGENE~~~'TC TCA CxTG C<1G ACA TGT~GGC T
'' "~-... .
TAG029GENE ~CAG GCT TCT GAT AGT TTC~ CA.~i CT
2 .
3'TACr029G~NE'-~~TAT
~ CT ATT CAG AGA AAC T v
G
i TAG030f~FIJE:. 1~'C~ A-4T GCA TGT AGA ACxC~T _
2.... _ __... .... . ...._
3 TAG030GEIr"E. ;~AGG GCA G
2 rAG TCG A
CA AAA CAG T~.~
!
i'TAGU82GEN'E_
2 . _
_
.. ~TCT TGA GTG GrGC TAG TGA ~CT
3'TAG032GENE A ~~ .. .
2 ".
~.__ zy__.~~__...__~, ~AGT CTG GC
.:. A ATCx
GAG ~CA~' G
_ _._. _.__._.._.._ ....
. _
i'TAG03fiGE?~TE,_2.,",.,._
_
_
.. _. ...
,...,..~'.~'-,rC_2AT,ACb TT~~~ACT~T'CA~T
i_TAGr03BG1~;IVE~;jTCC AAG TGT ACT GCT TAT
2
i'TAG03SGF.,r,N
'~' 2 I ~~CTA GTA G~.'C AGT ~'GG GAG T
~'TAG086GFnIITE,_8.1.~G'TC CAG AAC AGC CTT TAC T-y-'_ .
. ,: . .
CA 02369183 2002-O1-23
46
Table 8 Primer combinations and ampli&ed fragment length in the
Tag specific hTested PCR. reaetXOns
TAG004~~5' ' 3' TAG~004gene 2 182
TAG004gene-2
T , ~_..__.. 211._
46007 . j~, TA~7geue ~ , _...
~5' '
TAG007gene
~
1
"
T AGO10 _.. 108
. - ;y3' TAGOIOgene
, 2
~~5'
TAGOlOgene
~
.
T4G011-,_f~5' _ 239
TAGOllgene 3' TAG011~ene Z .
'? . .
'
~
_ _
;'TAG012.... ..- .
-. _. -I~-,-~AG012 e:~e-~ :.i97
._-_ ._ ..: _. .... _. .
. .z~~ _,_ ....... g ._...._-. .
y;AGfl1 ....- ,.... _. ..
~-- .
ene-2
..
..
-.
....".
.
..
.
.
...
~
.__..
. . _ _
'TAG013. 3 TAG013~g:~e 2 _ _
. . 212
. ~
=
~,5'
TAGU13g8na
2
,TAG014~ _ ~ 243
~' TAG~014~ene,~3' TAG014gene 2.
2 .
~
TAG01 ~.' ~.?' 'T'AGOhgene~3' TAG015gene-2 131
2
5
~TAGOlfiVia' . 3 TAG016gene-2 X19
t ..._TAGOIfi~ene .
2 .
_ .
_ . ....
:fTAG017... ",.. . . .. 185
_ 3' T~1G01?gene 2 '
.
._...
....
~A
TAG017gena
2
'T~G018-, . ' s TAG018gene ~
~5' ..
TAG018gerne ~
~
.
TAG019~~r' 3' TAG019gene 2 ~ ?04 -
TAG019gene .
~
.
;,TAG022, 3' TAGO~Zgene ~ ~ 2381
;~6'
TAG02~gene
2
'I'AG025,a5' 3' T4Cx02ogene 2 ._ ~83. .
TAG025gene
2
.
'TAG0~9_ 3' TAG029gene 2 .. _ __ 141
~e, . .. .
TA~29_gene : .
2 '
'
~T~G030... :. 179
. ~~ 's1 TAGOSOgezxe-~
~A~sOgene .. .
~
~'AG08~'~ ,,3~ TAG032gene 2 223
TAG032~erie
2
'I'AG036..... ._ :3..T~~'036gene .191 ,
- .. 2, ~, -~.. .... ...
J~~.T'~~036~ene~2... .
....
~tesults
The results of determination of the expression gavels of the genes identified
by
the ta.g sequences are depicted in ~.gure I9. ~'he data clearly indicate that
a
number of the genes identi~.ed by the tag sequences have a higher expression
IO in the skin samples W th Kaposi's Sarcoma lesions compared to normal skin:
tag097, tag010, tag012, tag013, tag014,, tag4l~, tagOlF,, tag017, tag022,
tag029, tag~30, tag032 and tag036.
CA 02369183 2002-O1-23
47
Example 11
Determination of the gene expression levels of the tae sequences in,peripheral
Mood mononuclear cell (1'B1VIC~sa~mples
To get a feeling for the use of the tag sequences as markers far angiogenesis
process in samples not fxoxn the location of the angiogenesis process, i_e.
the
Kaposi's Sarcoma in the skin or another tumour in the body but at an
accessible sample from the blood (Pf3MC's) tf.e expression. of 5 tag idanti~ed
genes vcras deterxrxined in PBMC samples: The PBMC samples were from the
blood of patients with {4 different samples) and without (2 control samples)
Kaposi's Saxcama lesions and were analysed for the expression level of 5 tag
identified genes i.n PEMC's.
The procedure of the e:~ample was identical as described in e:~ample 10, with
the exception that PBMC samples were used (approxi.mately 1.0 million cells
per sample, ranging from 2.5 to 50 million) instead of skin biopsies. The
genes
that w ere analysed ors identified by tag007, tag017, tag010, tag013, tag015,
tagC?29 and tag032. The results of the analysis are depicted is figure 20. Zt
is
clear from these data that the elevated expression. i.n the tumour sites of
the
genes identified by tag015 {TIE 1} and tag032 {Salioadhesin ox Siglec 3) is
2o paralleled in the blood cell fraction, i.e. pBMC.
Res is
The data olearly shave that the over expression of the gene identified by
tag00'T
{lieratiu 14) in skin samples (sae example 5) is. not paralleled in blood, In
contrast; in the samples tested in this example the gene identified by tag007
is
riot expressed at all ire the blood coxapartm.ent. This shows that no tumour
cells
expressing tag00? are present in blood. As a consequence, measurement of
tag007 in the blood c4uld be a good indicator for the prese~xce of these
tumour
CA 02369183 2002-O1-23
48
cells irl the blood, and thus a marker for circulating cancer cells that can
cause
metastasis.
The up regulation of genes identified by tag015 (TIE 1) and tag032
(Saliodhesin or Siglec l:) in skin samples is clearly paralleled ix~. the
blood.
These two tags are higher expressed in blood from patients with tumours
compared to healthy individuals. This up-regulation is due to up-regulation of
expression in typical blood cells. The up-regulation cannot be due to the
presence of tumour cells that express tag015 and tag~32 in the blood, because
the absebce of expression of the gene identified by tagOCf7 in the blood shows
that no tumour cells are present in blood, as explained above. This zr~eans
that
measurement of expression of tagal5 andlor tag032 in the blood indicates the
presence of a tumour somewhere else in the body. Furthermore, measurement
of expression of genes identified by tag0l.~ andlor tag032 during anti-tumour
therapy andlor anti-angiogenesis therapy can be used to monitor the efficacy
of
this txeatxaent.
Cox~clusioras
The paralleled up-regulation of the genes identified with tag010 (TIE 1) and
tag032 (Salioadhesin or Siglec 1) in both the t~~maur and the lalood, enables
the
monitoring of the efficacy of a therapy aimed at decreasing the growth df a
tumour; in particular anti-angiogenic tumour treatment. This follows the
reasoning that if these two genes are markers in f$MG for blood vessel
formation in a tumour in another site in the body, these two markers in blood
will also decrease with the decrease of this blood vessel growth at the tumour
?a site.
The genes identified by tag007 (Keratin 14), tag015 ('~'~ 1) and tag032
(Salioadhesin ox Siglec 1) have different expression in the blood of patients
with a tumour eoxt~pared to normal individuals. Therefore these genes, in
particular the expression thereof, can be used to screen a population at risk
for
the presence o~tumour in individual members of that populatioxi.
CA 02369183 2002-O1-23
49
All the genes identified by tags in this study that have charged expression
levels comparing normal to tumour tissue, i.e. tag007, tag0z0, tag012, tag013,
tag014, tag0~3, tag~l6, tag017, tag022, tag029, tag030, tag032 and tag03G axe
encoding potential target molecules for therapeutic compound with anti-
s angiogenic effects applicable in tumour treatment, ancUor these genes encode
potential target molecules that can be potential target rnalecules for
therapeutic coxxlpouuds that stimulate the gxo~wtlx of blood vessel, for
instance
in the treatment of heart and coronary disease.
CA 02369183 2002-O1-23
grief desez~iptian of the dra.wixigs
Figure 1: Sequence invalved in angiogeuesis. A change of expression of this
sequence after a certain treatzn.ent indicates that said treatment is
effective.
This sequence is identical to an ES'I' sequence identified fronx human foetal
heart (GenBank acc. # A,127.7565 and others}, which in turn matches a
predicted exon on chromosome 19. A relation with angiagenesis has not been
described previously.
Figuxe 2-18: Sequex~ces which are identified by name and Genbank numbers
(NCBI database). Other identi.fcation can be fou~xd in tables 1-4 (Gnigene
numbers) that can be found in the SAGE databases of NCBI.
Figure 10. Mean expression levels and standard deviation depicted as log
dilution factor that still is positive starting with 500 ng.of total RhiA
isolated
Pram ~ skin samples vsrith liaposi's Sarcoma (light bars) and 2 control,
normal
skin samples (dark bars).
Figure 20. Mean e:~pxession levels and standard deviation depicted. as log
dilution factox that still is positive staxting with 500 b,g of total ~i,NA
isolated
from 4 PBMC samples of patients with ~aposi's Sarcoma (light bars) and 2
control, normal FBMC samples (dark bars):
2~
CA 02369183 2002-04-23
-S 1-
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: Amsterdam Support Diagnostics B.V.
(B) STREET: Twin-2 Building R-South
(C) CITY: Meibergredreef 59
(D) STATE/PROVINCE: 1105 Amsterdam
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(F) POSTAL CODE/ZIP:
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(I) TELEFAX:
(ii) TITLE OF INVENTION: Means and Methods for Treatment Evaluation
(iii) NUMBER OF SEQUENCES: 156
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Borden Ladner Gervais LLP
(B) STREET: 1100-100 Queen Street
(C) CITY: Ottawa
(D) PROVINCE: Ontario
(E) COUNTRY: CANADA
(F) POSTAL CODE: K1P 1J9
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Ver. 2.1
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: 2,369,183
(B) FILING DATE: 23-JAN-2002
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: EP 01200228.3
(B) FILING DATE: 23-JAN-2001
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Fritz, Joachim
(B) REGISTRATION NUMBER: 4173
(C) REFERENCE/DOCKET NUMBER: PAT 50969-1
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (613) 237-5160
(B) TELEFAX: (613) 787-3558
(2) INFORMATION FOR SEQ ID NO:1:
CA 02369183 2002-04-23
-52-
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: TAG sequence Hs171596
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
ccccagtcgg c 11
(2) INFORMATION FOR SEQ ID N0:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: TAG sequence
Hs171695
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:2:
ettgacatac c 11
(2) INFORMATION FOR SEQ ID N0:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: Sequence: TAG sequence
Hs82112
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:3:
catcacggat c 11
(2) INFORMATION FOR SEQ ID N0:4:
CA 02369183 2002-04-23
-53-
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: TAG sequence Hs78436
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:4:
ggccaaaggc c 11
(2) INFORMATION FOR SEQ ID N0:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: TAG sequence
Hs82237
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:5:
ttgcatatca g 11
(2) INFORMATION FOR SEQ ID N0:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: TAG sequence
Hs78824
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:6:
ccctgttcag c 11
(2) INFORMATION FOR SEQ ID N0:7:
CA 02369183 2002-04-23
-54-
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH:11 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: TAG sequence
Hs16530
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:7:
gatcaatcag t 11
(2) INFORMATION FOR SEQ ID N0:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH:11 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: TAG sequence
Hs898
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:8:
gagggtgcca a 11
(2) INFORMATION FOR SEQ ID N0:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH:11 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: TAG sequence
Hs99923
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:9:
taaacctgct g 11
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
CA 02369183 2002-04-23
-55-
(A) LENGTH:11 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: TAG sequence
Hs1420
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SQ ID NO:10:
gtggccagag g 11
(2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH:11 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: TAG sequence
Hs183
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
tctggcccag c 11
(2) INFORMATION FOR SEQ ID N0:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH:11 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: TAG sequence
Hs75066
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:12:
caggtcgcta c 11
(2) INFORMATION FOR SEQ ID N0:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH:11 bases
(B) TYPE: nucleic acid
CA 02369183 2002-04-23
-56-
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: TAG sequence
Hs112408
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:13:
gagcagcgcc c 11
(2) INFORMATION FOR SEQ ID N0:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH:11 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: TAG sequence
Hs76152
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:14:
acttattatg c 11
(2) INFORMATION FOR SEQ ID N0:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH:11 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: TAG sequence
Hs74649 and Hs288761
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:15:
caggcctggc c 11
(2) INFORMATION FOR SEQ ID N0:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH:11 bases
(B) TYPE: nucleic acid
CA 02369183 2002-04-23
-57-
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: TAG sequence
Hs181062
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:16:
gtgcggagga c 11
(2) INFORMATION FOR SEQ ID N0:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH:11 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: TAG sequence
Hs74316
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:17:
acagcggcaa t 11
(2) INFORMATION FOR SEQ ID N0:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH:11 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: TAG sequence
Hs117729
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:18:
gatgtgcacg a 11
(2) INFORMATION FOR SEQ ID N0:19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH:11 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
CA 02369183 2002-04-23
-58-
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: TAG sequence
Hs24395
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:19:
caggtttcat a 11
(2) INFORMATION FOR SEQ ID N0:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH:11 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: TAG sequence
Hs93675
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:20:
aactctgacc c 11
(2) INFORMATION FOR SEQ ID N0:21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH:11 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: TAG sequence
Hs94953
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:21:
aaatcaatac a 11
(2) INFORMATION FOR SEQ ID N0:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH:11 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
CA 02369183 2002-04-23
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(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: TAG sequence
Hs108741
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:22:
tggtaactgg c 11
(2) INFORMATION FOR SEQ ID N0:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH:11 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: TAG sequence
Hs173789
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:23:
tctgcactga g 11
(2) INFORMATION FOR SEQ ID N0:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH:11 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: TAG sequence
Hs60440
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:24:
caggctgctg g 11
(2) INFORMATION FOR SEQ ID N0:25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH:11 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
CA 02369183 2002-04-23
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(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: TAG sequence
Hs13775
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:25:
atgacagatg g 11
(2) INFORMATION FOR SEQ ID N0:26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH:11 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: TAG sequence
Hs236510
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:26:
gcacaacaag a 11
(2) INFORMATION FOR SEQ ID N0:27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH:11 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: TAG sequence
Hs23579
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:27:
ccacaggaga a 11
(2) INFORMATION FOR SEQ ID N0:28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH:11 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
CA 02369183 2002-04-23
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(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: TAG sequence
Hs46987
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:28:
ctgtgcggaa c 11
(2) INFORMATION FOR SEQ ID N0:29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH:11 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: TAG sequence
Hs18104
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:29:
gatggctgcc t 11
(2) INFORMATION FOR SEQ ID N0:30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH:11 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: TAG sequence
Hs31869
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:30:
ctccattgcc a 11
(2) INFORMATION FOR SEQ ID N0:31:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH:11 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
CA 02369183 2002-04-23
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(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: TAG sequence
Hs112457
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:31:
acctccactg g 11
(2) INFORMATION FOR SEQ ID N0:32:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 46 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: oligo (dT)
primer with a 5' M13 tail
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:32:
ctagttgtaa aacgacggcc agtttttttt tttttttttt tttttt 46
(2) INFORMATION FOR SEQ ID N0:33:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: -21M13 primer
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:33:
gtaaaacgac ggccagt 17
(2) INFORMATION FOR SEQ ID N0:34:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
CA 02369183 2002-04-23
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(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: primer
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:34:
aaaaacatga cctccactgg 20
(2) INFORMATION FOR SEQ ID N0:35:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: TAG007
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:35:
aaaaacatgg atgtgcacg 19
(2) INFORMATION FOR SEQ ID N0:36:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: TAGO10
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:36:
aaaaacatgc cccagtcggc 20
(2) INFORMATION FOR SEQ ID N0:37:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: TAGO11
CA 02369183 2002-04-23
-64-
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:37:
aaaaacatgc ttgacatacc 20
(2) INFORMATION FOR SEQ ID N0:38:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: TAG012
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:38:
aaaaacatgc atcacggatc 20
(2) INFORMATION FOR SEQ ID N0:39:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: TAG013
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:39:
aaaaacatgg gccaaaggcc 20
(2) INFORMATION FOR SEQ ID N0:40:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: TAG014
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:40:
aaaaacatgt tgcatatcag 20
CA 02369183 2002-04-23
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(2) INFORMATION FOR SEQ ID N0:41:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
( ix) FEATURE
(A) NAME/KEY: Artificial Sequence: TAG015
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:41:
aaaaacatgc cctgttcagc 20
(2) INFORMATION FOR SEQ ID N0:42:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: TAG016
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:42:
aaaaacatgg atcaatcagt 20
(2) INFORMATION FOR SEQ ID N0:43:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: TAG017
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:43:
aaaaacatgg agggtgccaa 20
(2) INFORMATION FOR SEQ ID N0:44:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 bases
CA 02369183 2002-04-23
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(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: TAG018
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:44:
aaaaacatgt aaacctgctg 20
(2) INFORMATION FOR SEQ ID N0:45:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: TAG019
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:45:
aaaaacatgg tggccagagg 20
(2) INFORMATION FOR SEQ ID N0:46:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: TAG020
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:46:
aaaaacatgt ctggcccagc 20
(2) INFORMATION FOR SEQ ID N0:47:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
CA 02369183 2002-04-23
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(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: TAG021
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:47:
aaaaacatgc aggtcgctac 20
(2) INFORMATION FOR SEQ ID N0:48:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: TAG022
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:48:
aaaaacatgg agcagcgccc 20
(2) INFORMATION FOR SEQ ID N0:49:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: TAG033
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:49:
aaaaacatga cttattatgc 20
(2) INFORMATION FOR SEQ ID N0:50:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: TAG034
CA 02369183 2002-04-23
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(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:50:
aaaaacatgc aggcctggcc 20
(2) INFORMATION FOR SEQ ID N0:51:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: TAG035
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:51:
aaaaacatgg tgcggaggac 20
(2) INFORMATION FOR SEQ ID N0:52:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: TAG036
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:52:
aaaaacatga cagcggcaat 20
(2) INFORMATION FOR SEQ ID N0:53:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: TAG037
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:53:
CA 02369183 2002-04-23
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aaaaacatgc aggtttcata 20
(2) INFORMATION FOR SEQ ID N0:54:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: TAG038
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:54:
aaaaacatga actctgaccc 20
(2) INFORMATION FOR SEQ ID N0:55:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: TAG023
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:55:
aaaaacatga aatcaataca 20
(2) INFORMATION FOR SEQ ID N0:56:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: TAG024
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:56:
aaaaacatgt ggtaactggc 20
(2) INFORMATION FOR SEQ ID N0:57:
CA 02369183 2002-04-23
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: TAG025
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:57:
aaaaacatgt ctgcactgag 20
(2) INFORMATION FOR SEQ ID N0:58:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: TAG026
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:58:
aaaaacatgc aggctgctgg 20
(2) INFORMATION FOR SEQ ID N0:59:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: TAG027
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:59:
aaaaacatga tgacagatgg 20
(2) INFORMATION FOR SEQ ID N0:60:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
CA 02369183 2002-04-23
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(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: TAG028
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:60:
aaaaacatgg cacaacaaga 20
(2) INFORMATION FOR SEQ ID N0:61:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
( ix) FEATURE
(A) NAME/KEY: Artificial Sequence: TAG029
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:61:
aaaaacatgc cacaggagaa 20
(2) INFORMATION FOR SEQ ID N0:62:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: TAG030
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:62:
aaaaacatgc tgtgcggaac 20
(2) INFORMATION FOR SEQ ID N0:63:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
CA 02369183 2002-04-23
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(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: TAG031
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:63:
aaaaacatgg atggctgcct 20
(2) INFORMATION FOR SEQ ID N0:64:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: TAG032
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:64:
aaaaacatgc tccattgcca 20
(2) INFORMATION FOR SEQ ID N0:65:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 102 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
( ix) FEATURE
(A) NAME/KEY: Artificial Sequence: TAG004 (EST
AI217565, genbank number BE466728)
(B) LOCATION:
catgacctcc actggaagag ggggctagcg tgagcgctga ttctcaacct accataactc 60
tttcctgcct caggaactcc aataaaacat tttccatcca ac 102
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:65:
(2) INFORMATION FOR SEQ ID N0:66:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 242 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
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(A) NAME/KEY: Artificial Sequence: TAG007
(keratin 14, genbank number XM_008578)
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:66:
catggatgtg cacgatggca aggtggtgtc cacccacgag caggtccttc gcaccaagaa 60
ctgaggctgc ccagccccgc tcaggcctag gaggcccccc gtgtggacac agatcccact 120
ggaagatccc ctctcctgcc caagcacttc acagctggac cctgcttcac cctcaccccc 180
tcctggcaat caatacagct tcattatctg agttgctaaa aaaaaaaaaa aaaaaaaaaa 240
as 242
(2) INFORMATION FOR SEQ ID N0:67:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 240 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: TAGO10 (ephrin
A2, genbank number XM_002088)
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:67:
atctaccagc tcatgatgca gtgctggcag caggagcgtg cccaccgccc caagttcgct 60
gacatcgtca gcatcctgga caagctcatt cgtgcccctg actccctcaa gaccctggct 120
gactttgacc cccgcgtgtc tatccggctc cccagcacga gcggctcgga gggggtgccc 180
ttccgcacgg tgtccgagtg gctggagtcc atcaagatgc agcagtatac ggagcacttc 240
(2) INFORMATION FOR SEQ ID N0:68:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 355 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: TAGO11 (dual
specificity phosphatase, genbank number XM_003720)
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:68:
catgcttgac atacctacca gtattattcc cgacgacaca tatacatatg agaatatacc 60
ttatttattt ttgtgtaggt gtctgccttc acaaatgtca ttgtctactc ctagaagaac 120
caaatacctc aatttttgtt tttgagtact gtactatcct gtaaatatat cttaagcagg 180
tttgttttca gcactgatgg aaaataccag tgttgggttt ttttttagtt gccaacagtt 240
gtatgtttgc tgattattta tgacctgaaa taatatattt cttcttctaa gaagacattt 300
tgttacataa ggatgacttt tttatacaat ggaataaatt atggcatttc tattg 355
CA 02369183 2002-04-23
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(2} INFORMATION FOR SEQ ID N0:69:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 63 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: TAG012 (IL1
receptor, type 1, genbank number XM-002686)
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:69:
catgcatcac ggatcaatag actgtactta ttttccaata aaattttcaa actttgtact 60
gtt 63
(2) INFORMATION FOR SEQ ID N0:70:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 212 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: TAG013 (ephrin
B1, genbank numbers XM_002535, BC002524)
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:70:
aacttgccct gtgcctgtgt cccccatgct aggggcggag gggtcttttc cttcttcttt 60
cctacctacc ccttttctct tggccagggg cctcgtatcc tacctttcct tgtcccctgg 120
gctggctgca cagaggattg ccccttctct tttcagagct ggccctcgat gccaaattag 180
catttagtat tttgctcaaa gtctaaggga cc 212
(2) INFORMATION FOR SEQ ID N0:71:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 214 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: TAG014 (AT
group D protein, genbank numbers XM-006184,
AF230388)
(B) LOCATION:
CA 02369183 2002-04-23
-75-
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:71:
catgttgcat atcagggtgc tcaaggattg gagaggagac aaaaccagga gcagcacagt 60
ggggacatct cccgtctcaa cagccccagg cctatggggg ctctggaagg atgggccagc 120
ttgcaggggt tggggaggga gacatccagc ttgggctttc ccctttggaa taaaccattg 180
gtctgtcaca aaaaaaaaaa aaaaaaaaaa aaaa 214
(2) INFORMATION FOR SEQ ID N0:72:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 93 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: TAG015 (TIE l,
genbank number XM_002037)
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:72:
catgccctgt tcagctactc ccactcccgg cctgtcattc agaaaaaaat aaatgttcta 60
ataagctcca aaaaaaaaaa aaaaaaaaaa aaa 93
(2) INFORMATION FOR SEQ ID N0:73:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 163 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: TAG016 (small
ind. Cytokine A18, genbank numbers XM-008451,
Y13710, AF111198)
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:73:
catggatcaa tcagtgtgat tagctttctc agcagacatt gtgccatatg tatcaaatga 60
caaatcttta ttgaatggtt ttgctcagca ccacctttta atatattggc agtacttatt 120
atataaaagg taaaccagca ttctcaaaaa aaaaaaaaaa aaa 163
(2) INFORMATION FOR SEQ ID N0:74:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 205 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
CA 02369183 2002-04-23
-76-
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: TAG017
(complement comp. 1Q beta, genbank number
XM_010666)
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:74:
catggagggt gccaacagca tcttttccgg gttcctgctc tttccagata tggaggcctg 60
acctgtgggc tgcttcacat ccaccccggc tccccctgcc agcaacgctc actctacccc 120
caacaccacc ccttgcccag ccaatgcaca cagtagggct tggtgaatgc tgctgagtga 180
atgagtaaat aaactcttca aggcc 205
(2) INFORMATION FOR SEQ ID N0:75:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 175 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: TAG018
(galectin 7, genbank numbers NM_002307, U06643)
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:75:
cggctggaca cgtcggaggt ggtcttcaac agcaaggagc aaggctcctg gggccgcgag 60
gagcgcgggc cgggcgttcc tttccagcgc gggcagccct tcgaggtgct catcatcgcg 120
tcagacgacg gcttcaaggc cgtggttggg gacgcccagt accaccactt ccgcc 175
(2) INFORMATION FOR SEQ ID N0:76:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 204 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: TAG019 (FGFR3,
genbank numbers NM-022965, NM-000142)
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:76:
cacaacctcg actactacaa gaagacaacc aacggccggc tgcccgtgaa gtggatggcg 60
cctgaggcat tatttgaccg agtctacact caccagagtg acgtctggtc ctttggggtc 120
ctgctctggg agatcttcac gctggggggc tccccgtacc ccggcatccc tgtggaggag 180
ctcttcaagc tgctgaagga gggc 204
(2) INFORMATION FOR SEQ ID N0:77:
CA 02369183 2002-04-23
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 95 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: TAG022
(Psoriasin (5100 A7), genbank number XM_048120)
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:77:
catggagcag cgccctgttc cgggggcagc cagtgaccca gccccaccaa tgggcctcca 60
gagaccccag gaacaataaa atgtcttctc ccacc 95
(2) INFORMATION FOR SEQ ID N0:78:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 139 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: TAG025 (EST
Unigene no. Hs173789, genbank numbers XM-018404,
AL137262)
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:78:
catgtctgca ctgagaaact gcatttcagt agcatttgtc atccagccgg aagttaaagc 60
acacttactt tattcaccta tttttataat aaacgttctt gctgctgtga aaaaaaaaaa 120
aaaaaaaaaa aaaaaaaaa 139
(2) INFORMATION FOR SEQ ID N0:79:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH:234 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: TAG029 (PIG,
genbank numbers XM-011453, AJ251830)
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:79:
CA 02369183 2002-04-23
_78_
catgccacag gagaattcgg ggatttgagt ttctctgaat agcatatata tgatgcatcg 60
gataggtcat tatgattttt taccatttcg acttacataa tgaaaaccaa ttcattttaa 120
atatcagatt attattttgt aagttgtgga aaaagctaat tgtagttttc attatgaagt 180
tttcccaata aaccaggtat tctaaacttg aaaaaaaaaa aaaaaaaaaa aaaa 234
(2) INFORMATION FOR SEQ ID N0:80:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 194 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: TAG030 (EST
Unigene no. Hs46987, genbank numbers DG151190,
BG057289, BE858276, AV681759, BE503169)
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:80:
catgctgtgc ggaactgcgt cagggcaaat gtcacagcag gatttcccca acccagctcc 60
atcatcacag acacagaggg ctgcagggga ggcctgccca ctgttttgtc gactctgccc 120
tcctctggca gcatagatcc ttaggtgctc aataaaggtg tgctgtattg aaaaaaaaaa 180
aaaaaaaaaa aaaa 194
(2) INFORMATION FOR SEQ ID N0:81:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 97 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: TAG032
(SialoAdhesin, also called Siglec 1, genbank
number XM_016245)
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:81:
catgctccat tgccagactc ttgctgggag cccgtccaga atgtcctccc aataaaactc 60
catcctatga cgcaaaaaaa aaaaaaaaaa aaaaaaa 97
(2) INFORMATION FOR SEQ ID N0:82:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 143 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
CA 02369183 2002-04-23
-79-
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: TAG036
(Desmoplakin, genbank numbers XM_004463,
NM_004415, AF139065)
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:82:
catgacagcg gcaatctttt ctttggtcaa agttttctgt ttattttgct tgtcatattc 60
gatgtacttt aaggtgtctt tatgaagttt gctattctgg caataaactt ttagacttta 120
aaaaaaaaaa aaaaaaaaaa aaa 143
(2) INFORMATION FOR SEQ ID N0:83:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/FtEY: Artificial Sequence: 5'TAG004GENE
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:83:
ggcctttaac accccgttcc t 21
(2) INFORMATION FOR SEQ ID N0:84:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: 3'TAG004GENE
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:84:
tggtaggttg agaatcagcg ctca 24
(2) INFORMATION FOR SEQ ID N0:85:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 bases
(B)~TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
CA 02369183 2002-04-23
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: 5'TAG007GENE-N
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:85:
aggagaccaa aggtcgctac tgca 24
(2) INFORMATION FOR SEQ ID N0:86:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: 3'TAG007GENE
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:86:
cagttcttgg tgcgaaggac ct 22
(2) INFORMATION FOR SEQ ID N0:87:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: 5'TAGOlOGENE
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:87:
atctaccagc tcatgatgca gtgct 25
(2) INFORMATION FOR SEQ ID N0:88:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
CA 02369183 2002-04-23
-81-
(A) NAME/KEY: Artificial Sequence: 3'TAGOlOGENE
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:88:
gaagtgctcc gtatactgct gcat 24
(2) INFORMATION FOR SEQ ID N0:89:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: 5'TAGO11GENE
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:89:
agtgggtaca tcaagtccat ctga 24
(2) INFORMATION FOR SEQ ID N0:90:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: 3'TAGO11GENE
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:90:
cactggtatt ttccatcagt get 23
(2) INFORMATION FOR SEQ ID N0:91:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: 5'TAG012GENE
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:91:
CA 02369183 2002-04-23
-82-
taaagttgtc ctgcttgagc tgga 24
(2) INFORMATION FOR SEQ ID N0:92:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: 3'TAG012GENE
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:92:
ggcacgtgag cctctctttg cagt 24
(2) INFORMATION FOR SEQ ID N0:93:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: 5'TAG013GENE
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:93:
ctctacccca gaggaattta caga 24
(2) INFORMATION FOR SEQ ID N0:94:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: 3'TAG013GENE
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:94:
gggccagacc aaacacagac ctct 24
CA 02369183 2002-04-23
-83-
(2) INFORMATION FOR SEQ ID N0:95:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: 5'TAG014GENE
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:95:
ggcaacaagc agaaggcggt ca 22
(2) INFORMATION FOR SEQ ID N0:96:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: 3'TAG014GENE
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:96:
tgatcttgag ctgcagctgc tcct 24
(2) INFORMATION FOR SEQ ID N0:97:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence:
(B) LOCATION: 5'TAG015GENE
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:97:
gaatgtgctg gtcggagaga a 21
(2) INFORMATION FOR SEQ ID N0:98:
(i) SEQUENCE CHARACTERISTICS:
CA 02369183 2002-04-23
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(A) LENGTH: 22 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: 3'TAG015GENE
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:98:
tggggcagct tttcatagag ct 22
(2) INFORMATION FOR SEQ ID N0:99:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: 5'TAG016GENE
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:99:
ttctctgcct gcccagcatc atga 24
(2) INFORMATION FOR SEQ ID NO:100:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: 3'TAG016GENE
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:100:
tcaggcattc agcttcaggt cgct 24
(2) INFORMATION FOR SEQ ID NO:101:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
CA 02369183 2002-04-23
-85-
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: 5'TAG017GENE
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:101:
gtctctacta cttcacctac ca 22
(2) INFORMATION FOR SEQ ID N0:102:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
( ix) FEATURE
(A) NAME/KEY: Artificial Sequence: 3'TAG017GENE
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:102:
tgttgggggt agagtgagcg ttgct 25
(2) INFORMATION FOR SEQ ID N0:103:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: 5'TAG018GENE
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:103:
gcaggttcca tgtaaacctg ctgt 24
(2) INFORMATION FOR SEQ ID N0:104:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: 3'TAG018GENE
CA 02369183 2002-04-23
-86-
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:104:
ctgctcagaa gatcctcacg gagt 24
(2) INFORMATION FOR SEQ ID N0:105:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: 5'TAG019GENE
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:105:
gtgaccgagg acaacgtgat gaaga 25
(2) INFORMATION FOR SEQ ID N0:106:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: 3'TAG019GENE
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:106:
catgatcatg tacaggtcgt gtgt 24
(2) INFORMATION FOR SEQ ID N0:107:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: 5'TAG022GENE
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:107:
tgagcaacac tcaagctgag ag 22
CA 02369183 2002-04-23
_g7_
(2) INFORMATION FOR SEQ ID N0:108:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: 3'TAG022GENE
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:108:
tctctggagg cccattggt 19
(2) INFORMATION FOR SEQ ID N0:109:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: 5'TAG025GENE
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:109:
atggggtcag gaacatctgg caga 24
(2) INFORMATION FOR SEQ ID N0:110:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 bases
(B) TYPE: nucleic acid
(C) STR.ANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: 3'TAG025GENE
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:110:
tccggctgga tgacaaatgc tact 24
(2) INFORMATION FOR SEQ ID NO:111:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 bases
CA 02369183 2002-04-23
_8g_
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: 5'TAG029GENE
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:111:
ctcaggttta tctgggctct atca 24
(2) INFORMATION FOR SEQ ID N0:112:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: 3'TAG029GENE
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:112:
tcataatgac ctatccgatg cat 23
(2) INFORMATION FOR SEQ ID N0:113:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: 5'TAG030GENE
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:113:
cttgcaaaga taggagaggc tcca 24
(2) INFORMATION FOR SEQ ID N0:114:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
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(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: 3'TAG030GENE
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:114:
attgagcacc taaggatcta tgct 24
(2) INFORMATION FOR SEQ ID N0:115:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: 5'TAG032GENE
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:115:
tgcgaatcag ggaccaacag gaga 24
(2) INFORMATION FOR SEQ ID N0:116:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: 3'TAG032GENE
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:116:
ttgggaggac attctggacg ggct 24
(2) INFORMATION FOR SEQ ID N0:117:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: 5'TAG036GENE
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(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:117:
atttagcagt agttctattg ggca 24
(2) INFORMATION FOR SEQ ID N0:118:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: 3'TAG036GENE
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:118:
actgattagc acttcagacg cact 24
(2) INFORMATION FOR SEQ ID N0:119:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: 5'TAG004GENE-2
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:119:
catcgacaaa ttgcgatct 19
(2) INFORMATION FOR SEQ ID N0:120:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: 3'TAG004GENE-2
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:120:
cgctagcccc ctcttccagt 20
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(2) INFORMATION FOR SEQ ID N0:121:
(1) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: 5'TAG007GENE-2.1
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:121:
aggagatgat tggcagcgt 19
(2) INFORMATION FOR SEQ ID N0:122:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(1i) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: 3'TAG007GENE-2
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:122:
ggaggaggtc acatctctgg at 22
(2) INFORMATION FOR SEQ ID N0:123:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: 5'TAGOlOGENE-2
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:123:
ccaagttcgc tgacatcgt 19
(2) INFORMATION FOR SEQ ID N0:124:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 bases
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(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: 3'TAGOlOGENE-2
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:124:
tgctggggag ccggatagac a 21
(2) INFORMATION FOR SEQ ID N0:125:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: 5'TAGO11GENE-2
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:125:
gaagagaaag gactcagtgt 20
(2) INFORMATION FOR SEQ ID N0:126:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: 3'TAG011GENE-2
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:126:
agatatattt acaggatagt 20
(2) INFORMATION FOR SEQ ID N0:127:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
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(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: 5'TAG012GENE-2
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:127:
aaatccaaga ctatgaga 18
(2) INFORMATION FOR SEQ ID N0:128:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: 3'TAG012GENE-2
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:128:
cttagtggct ggtgacagt 19
(2) INFORMATION FOR SEQ ID N0:129:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: 5'TAG013GENE-2
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:129:
aacttgccct gtgcctgtgt 20
(2) INFORMATION FOR SEQ ID N0:130:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: 3'TAG013GENE-2
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(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:130:
ggtcccttag actttgagca 20
(2) INFORMATION FOR SEQ ID N0:131:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: 5'TAG014GENE-2
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:131:
cttctgcgag ctgcatctca 20
(2) INFORMATION FOR SEQ ID N0:132:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: 3'TAG014GENE-2
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:132:
tgcagtgaca gctccgtct 19
(2) INFORMATION FOR SEQ ID N0:133:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: 5'TAG015GENE-2
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:133:
agaggaggtt tatgtgaaga 20
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(2) INFORMATION FOR SEQ ID N0:134:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: 3'TAG015GENE-2
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:134:
actatctccc aaagaaggac t 21
(2) INFORMATION FOR SEQ ID N0:135:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: 5'TAG016GENE-2
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:135:
tgtcctcgtc tgcaccat 1g
(2) INFORMATION FOR SEQ ID N0:136:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: 3'TAG016GENE-
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:136:
atgtatttct ggacccact
19
(2) INFORMATION FOR SEQ ID N0:137:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: bases
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(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: 5'TAG017GENE-2
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:137:
gtcaccttct gtgactatgc ct 22
(2) INFORMATION FOR SEQ ID N0:138:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: 3'TAG017GENE-2
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:138:
acaggtcagg cctccatatc t 21
(2) INFORMATION FOR SEQ ID N0:139:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: 5'TAG018GENE-2
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:139:
cggctggaca cgtcgga 17
(2) INFORMATION FOR SEQ ID N0:140:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
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(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/ICEY: Artificial Sequence: 3'TAG018GENE-2
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:140:
ggcggaagtg gtggtact i8
(2) INFORMATION FOR SEQ ID N0:141:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: 5'TAG019GENE-2
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:141:
cacaacctcg actactaca 19
(2) INFORMATION FOR SEQ ID N0:142:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: 3'TAG019GENE-2
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:142:
gccctccttc agcagctt 18
(2) INFORMATION FOR SEQ ID N0:143:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: 5'TAG022GENE-2
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(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:143:
ttcacaaata caccagacgt gat 23
(2) INFORMATION FOR SEQ ID N0:144:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: 3'TAG022GENE-2
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:144:
gggcgctgct ccatggctct get 23
(2) INFORMATION FOR SEQ ID N0:145:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: 5'TAG025GENE-2
(B) LOCATION:
(x1) SEQUENCE DESCRIPTION: SEQ ID N0:145:
tgcctagaaa ggggtggct 19
(2) INFORMATION FOR SEQ ID N0:146:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: 3'TAG025GENE-2
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:146:
ttctcagtgc agacatgtgg ct 22
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(2) INFORMATION FOR SEQ ID N0:147:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: 5'TAG029GENE-2
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:147:
caggcttctg atagtttgca act 23
(2) INFORMATION FOR SEQ ID N0:148:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: 3'TAG029GENE-2
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:148:
tatgctattc agagaaact 19
(2) INFORMATION FOR SEQ ID N0:149:
(1) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: 5'TAG030GENE-2
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:149:
tctaatgcat gtagaagct 19
(2) INFORMATION FOR SEQ ID N0:150:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 bases
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(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: 3'TAG030GENE-2
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:150:
agggcagagt cgacaaaaca gt 22
(2) INFORMATION FOR SEQ ID N0:151:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: 5'TAG032GENE-2
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:151:
tcttgagtgg gctagtgact 20
(2) INFORMATION FOR SEQ ID N0:152:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: 3'TAG032GENE-2
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:152:
agtctggcaa tggagcatga 20
(2) INFORMATION FOR SEQ ID N0:153:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
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(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: 5'TAG036GENE-2
(B) LOCATION:
(x1) SEQUENCE DESCRIPTION: SEQ ID N0:153:
tgctatacct tgacttcat 19
(2) INFORMATION FOR SEQ ID N0:154:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(11) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: 3'TAG036GENE-2
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:154:
tccaagtgta ctgcttat 18
(2) INFORMATION FOR SEQ ID N0:155:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: 5'TAG036GENE-2.1
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:155:
ctagtagtca gttgggagt 19
(2) INFORMATION FOR SEQ ID N0:156:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Artificial Sequence: 3'TAG036GENE-2.1
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(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:156:
agccagaaca gcctttact 19
