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NUCLEIC ACID ARRAYS
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a continuation-in-part of application serial no.
08/859,998 filed on
May 21, 1997 and application serial no. 09/053,375 filed on March 31, 1998,
the disclosures
of which are herein incorporated by reference.
INTRODUCTION
Technical Field
The field of this invention is biopolymeric arrays.
Background of the Invention
"Biochips" or arrays of binding agents, such as oiigonucleotides and peptides,
have
become an increasingly important tool in the biotechnology industry and
related fields.
I S These binding agent arrays, in which a plurality of binding agents are
deposited onto a solid
support surface in the form of an array or pattern, find use in a variety of
applications,
including drug screening, nucleic acid sequencing, mutation analysis, and the
like. One
important use of biochips is in the analysis of differential gene expression,
where the
expression of genes in different cells, normally a cell of interest and a
control, is compared
and any discrepancies in expression are identified. In such assays, the
presence of
discrepancies indicates a difference in the classes of genes expressed in the
cells being
compared.
In methods of differential gene expression, arrays find use by serving as a
substrate to
which is bound polynucleotide "probe" fragments. One then obtains "targets"
from
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analogous cells, tissues or organs of a healthy and diseased organism. The
targets are then
hybridized to the immobilized set of polynucleotide "probe" fragments.
Differences between
the resultant hybridization patterns are then detected and related to
differences in gene
expression in the two sources.
A variety of different array technologies have been developed in order to meet
the
growing need of the biotechnology industry, as evidenced by the extensive
number of
patents and references listed in the relevant literature section below.
Despite the wide variety of array technologies currently in preparation or
available on
the market, there is a continued need to identify new array devices to meet
the needs of
specific applications. Of particular interest would be the development of an
array capable of
providing high throughput analysis of differential gene expression.
Relevant Literature
Patents and patent applications describing arrays of biopolymeric compounds
and
methods for their fabrication include: 5,242,974; 5,384,261; 5,405,783;
5,412,087;
5,424,186; 5,429,807; 5,436,327; 5,445,934; 5,472,672; 5,527,681; 5,529,756;
5,545,531;
5,554,501; 5,556,752; 5,561,071; 5,599,895; 5,624,711; 5,639,603; 5,658,734;
WO
93/I7126; WO 95/11995; WO 95/35505; EP 742 287; and EP 799 897.
Patents and patent application describing methods of using arrays in various
applications include: 5,143,854; 5,288,644; 5,324,633; 5,432,049; 5,470,710;
5,492,806;
5,503,980; 5,510,270; 5,525,464; 5,547.839; 5,580,732; 5,661,028; WO 95/21265;
WO
96/31622; WO 97/10365; WO 97/27317; EP 373 203; and EP 785 280.
Other references of interest include: Atlas Human cDNA Expression Array I
(April
1997) CLONTECHniques XII: 4-7; Lockhart et al., Nature Biotechnology (1996)
14: 1675-
1680; Shena et al., Science (1995) 270: 467-470; Schena et al., Proc. Nat'1
Acad. Sci. USA
(1996)93:10614-10619; Shalon et al., Genome Res. (1996) 6: 639-645;
Milosavljevic et al.,
Genome Res. ( 1996) 6:132-141; Nguyen et al., Genomics ( I 995)29: 207-216;
Pietu et al.,
Genome Res. ( 1996) 6: 492-503; Zhao et al., Gene ( 1995) 166:207-213;
Chalifour et al.,
Anal. Biochem. (1994) 216:299-304; Heller et al., Proc. Nat'1 Acad. Sci. USA
(1997) 94:
2150-2155: and Schena, M., BioAssays ( 1996) 18: 427-431.
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SUMMQ~Y OF THE INVENTION
Arrays of polynucleotide spots stably associated with the surface of a solid
support
and kits comprising the same, as well as methods for their preparation and use
in
hybridization assays, are provided. The subject arrays comprise a plurality of
polynucleotide
spots, wherein each different polynucleotide spot is made up of a
polynucleotide probe
composition and at least a portion of the polynucleotide probe compositions
are made up of
unique polynucleotides. The arrays are further characterized in that all of
the unique
polynucleotides on the array correspond to the same type of gene. The subject
arrays find
particular use in differential gene expression analysis. Also provided are
sets of a
representational number of gene specific primers useful in generating target
nucleic acids for
use with the subject arrays in hybridization assays.
BRIEF DESCRIPTION OF THE FIGURES
Fig. 1 provides a representation of an array according to the subject
invention.
I5
DEFINITIONS
The term "nucleic acid" as used herein means a polymer composed of
nucleotides,
e.g. deoxyribonucleotides or ribonucleotides.
The terms "ribonucleic acid" and "RNA" as used herein mean a polymer composed
of ribonucleotides.
The terms "deoxyribonucleic acid" and "DNA" as used herein mean a polymer
composed of deoxyribonucleotides.
The term "oligonucleotide" as used herein denotes single stranded nucleotide
multimers of from about 10 to 100 nucleotides in length.
The term "polynucleotide" as used herein refers to single or double stranded
polymer
composed of nucleotide monomers of greater than about 120 nucleotides in
length up to
about 1000 nucleotides in length.
The term "array type" refers to the type of gene represented on the array by
the
unique polynucleotides, where the type of gene that is represented on the
array is dependent
on the intended purpose of the array, e.g. to monitor expression of key human
genes, to
monitor expression of known oncogenes, etc, i.e. the use for which the array
is designed. As
such, all of the unique polynucleotides on a given array correspond to the
same type or
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category or group of genes. Genes are considered to be of the same type if
they share some
common linking characteristics, such as: species of origin, e.g. human, mouse,
rat, etc.;
tissue or cell type of origin, e.g. muscle, neural, dermal, organ, etc.;
disease state, e.g. cancer;
functions, e.g. protein kinases, tumor supressors and the tike, participation
in the same
normal biological process, e.g. apoptosis, signal transduction, cell cycle
regulation,
proliferation, differentiation etc.; and the like. For example, one array type
that is provided
below is a ''cancer array" in which each of the "unique" poiynucleotide probes
correspond to
a gene associated with a cancer disease state. Likewise, a "human array" may
be an array of
polynucleotides corresponding to unique tightly regulated human genes.
Similarly, an
I O "apoptosis array" may be an array type in which the polynucleotides
correspond to unique
genes associated with apoptosis.
The "unique" polynucleotide sequences associated with each type of array of
the
present invention are sequences which are distinctive or different with
respect to every other
polynucleotide sequence on the array and correspond to the same type of gene,
as defined
above. For example, in a cancer array, each unique polynucleotide has a
sequence that is not
homologous to any other known cancer associated sequence. Moreover, each
polynucleotide
sequence on the array is statistically chosen to ensure that the probability
of homology to any
sequence of that type is very low. Morever, in the cancer array embodiment,
all sequences
are statistically chosen to insure that the probability of homology to any
other sequence
associated with cancer or of human origin is very low. An important feature of
the individual
polynucleotide probe compositions of the subject arrays is that they are only
a fragment of
the entire cDNA of the gene to which they correspond. In other words, for each
gene
represented on the array, the entire cDNA sequence the gene is not represented
on the array.
Instead, the sequence of only a portion or fragment of the entire cDNA is
represented on the
array by this unique polynucleotide.
The term "polynucleotide probe composition" refers to the nucleic acid
composition
that makes up each of the spots on the array. Thus, the term '' poiynucleotide
probe
composition" includes nucleic acid compositions of unique polynucleotides and
control or
calibrating polynucleotides (e.g. polynucleotides corresponding to
housekeeping genes). The
polynucleotide compositions are made up of single stranded polynucleotides
(i.e.
polynucleotides that are not hybridized to each other), where all of the
polynucleotides in the
probe composition may be identical to each other or there may be two different
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polynucleotides (polynucleotides of different nucleotide sequence) in each
probe
composition, where the two different polynucleotides are complementary to each
other.
The term "gene specific primer" means a polynucleotide of sufficient length to
specifically hybridize to a distinct nucleic acid member of the sample, e.g.
RNA or cDNA,
where the length of the gene specific primers will usually be at least 8 nt,
more usually at
least 20 nt and may be as long as 25 nt or longer, but will usually not exceed
50 nt. The gene
specific primers of the subject invention are sufficiently specific to
hybridize to
complementary template sequence during the generation of labeled nucleic acids
under
conditions sufficient for first strand cDNA synthesis, which conditions are
known by those
of skill in the art. The number of mismatches between the gene specific primer
sequences
and their complementary template sequences to which they hybridize during the
generation
of labeled nucleic acids in the subject methods will generally not exceed 20
%, usually will
not exceed 10 % and more usually will not exceed 5 %, as determined using the
FASTA
program using default settings.
DESCRIPTION OF THE SPECIFIC EMBODIMENTS
Arrays of polynucleotide spots and methods for their preparation are provided.
In the
subject arrays, a plurality of polynucleotide spots is stably associated with
the surface of a
solid support, where at least a portion of the polynucleotide spots on the
array are made up
of unique polynucleotides and all of the unique polynucleotides of the array
correspond to
one particular type of gene, e.g. tightly regulated human genes, genes
associated with a
particular disease state, genes associated with cell cycle regulation, etc.
The subject arrays
find particular use in gene expression assays. Also provided are sets of a
representational
number of gene specific primers useful in generating target nucleic acids for
use with the
subject arrays. In further describing the subject invention, the arrays first
will be described in
. general terms. Next, methods for their preparation are described. Following
this, a
description of representative specific array types falling within the scope of
the invention
will be provided. Finally, a review of representative applications in which
the subject arrays
may be employed will be provided, where this review includes a description of
the sets of a
representational number of gene specitic primers according to the subject
invention.
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Before the subject invention is further described, it is to be understood that
the
invention is not limited to the particular embodiments of the invention
described below, as
variations of the particular embodiments may be made and still fall within the
scope of the
appended claims. It is also to be understood that the terminology employed is
for the purpose
of describing particular embodiments, and is not intended to be limiting.
Instead, the scope
of the present invention will be established by the appended claims.
In this specification and the appended claims, the singular forms "a," "an,"
and "the"
include plural reference unless the context clearly dictates otherwise. Unless
defined
otherwise, all technical and scientific terms used herein have the same
meaning as
commonly understood to one of ordinary skill in the art to which this
invention belongs.
ARRAYS OF THE SUBJECT INVENTION-GENERAL DESCRIPTION
1 S Array Structure
The arrays of the subject invention have a plurality of polynucleotide spots
stably
associated with a surface of a solid support. Each spot on the array comprises
a
polynucleotide sample, i.e. polynucleotide probe composition, of known
identity, usually of
known sequence, as described in greater detail below. The polynucleotide spots
on the array
may be any convenient shape, but will typically be circular, elliptoid, oval
or some other
analogously curved shape. The density of the spots on the solid surface is at
least about
S/cm= and usually at least about 10/cm= but does not exceed about 1000/cm'-,
and usually
does not exceed about 500/cm'-, and more usually does not exceed about 300/cm'-
. The spots
may be arranged in any convenient pattern across or over the surface of the
array, such as in
rows and columns so as to form a grid, in a circular pattern, and the like,
where generally the
pattern of spots will be present in the form of a grid across the surface of
the solid support.
See Fig. 1.
In the subject arrays, the spots of the pattern are stably associated with the
surface of
a solid support, where the support may be a flexible or rigid solid support.
By stably
associated is meant that the polynucleotides of the spots maintain their
position relative to
the solid support under hybridization and washing conditions. As such, the
polynucleotide
members which make up the spots can be non-covalently or covalently stably
associated
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with the support surface. Examples of non-covalent association include non-
specific
adsorption, binding based on electrostatic (e.g. ion, ion pair interactions),
hydrophobic
interactions, hydrogen bonding interactions, specific binding through a
specific binding pair
member covalently attached to the support surface, and the like. Examples of
covalent
S binding include covalent bonds formed between the spot polynucleotides and a
functional
group present on the surface of the rigid support, e.g. -OH, where the
functional group may
be naturally occurring or present as a member of an introduced linking group,
as described in
greater detail below.
As mentioned above, the array is present on either a flexible or rigid
substrate. By
flexible is meant that the support is capable of being bent, folded or
similarly manipulated
without breakage. Examples of solid materials which are flexible solid
supports with respect
to the present invention include membranes, e.g. nylon, flexible plastic
films, and the like.
By rigid is meant that the support is solid and does not readily bend, i.e.
the support is not
flexible. As such, the rigid substrates of the subject arrays are sufficient
to provide physical
support and structure to the polymeric targets present thereon under the assay
conditions in
which the array is employed, particularly under high throughput handling
conditions.
Furthermore, when the rigid supports of the subject invention are bent, they
are prone to
breakage.
The solid supports upon which the subject patterns of spots are presented in
the
subject arrays may take a variety of configurations ranging from simple to
complex,
depending on the intended use of the array. Thus, the substrate could have an
overall slide or
plate configuration, such as a rectangular or disc configuration. In many
embodiments, the
substrate will have a rectangular cross-sectional shape, having a length of
from about 10 mm
to 200 mm, usually from about 40 to 150 mm and more usually from about 7~ to
125 mm
and a width of from about 10 mm to 200 mm, usually from about 20 mm to 120 mm
and
more usually from about 25 to 80 mm, and a thickness of from about 0.01 mm to
5.0 mm,
usually from about 0.1 mm to 2 mm and more usually from about 0.2 to 1 mm.
The substrates of the subject arrays may be fabricated from a variety of
materials.
The materials from which the substrate is fabricated should ideally exhibit a
low level of
non-specific binding during hybridization events. In many situations, it will
also be
preferable to employ a material that is transparent to visible and/or UV
light. For tlexible
substrates, materials of interest include: nylon, both modified and
unmodified, nitrocellulose,
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polypropylene, and the like, where a nylon membrane, as well as derivatives
thereof, is of
particular interest in this embodiment. For rigid substrates, specific
materials of interest
include: glass; plastics, e.g. polytetrafluoroethylene, polypropylene,
polystyrene,
polycarbonate, and blends thereof, and the like; metals, e.g. gold, platinum,
and the like; etc.
The substrates of the subject arrays comprise at least one surface on which
the pattern
of spots is present, where the surface may be smooth or substantially planar,
or have
irregularities, such as depressions or elevations. The surface on which the
pattern of spots is
present may be modified with one or more different layers of compounds that
serve to
modify the properties of the surface in a desirable manner. Such modification
layers, when
present, wilt generally range in thickness from a monomolecular thickness to
about 1 mm,
usually from a monomolecular thickness to about 0.1 mm and more usually from a
monomolecular thickness to about 0.001 mm. Modification layers of interest
include:
inorganic and organic layers such as metals, metal oxides, polymers, small
organic
molecules and the like. Polymeric layers of interest include layers of:
peptides, proteins,
polynucleic acids or mimetics thereof, e.g. peptide nucleic acids and the
like;
polysaccharides, phospholipids, polyurethanes, polyesters, polycarbonates,
polyureas,
polyamides, polyethyleneamines, polyarylene sulfides, poIysiloxanes,
polyimides,
polyacetates, and the like, where the polymers may be hetero- or
homopolymeric, and may
or may not have separate functional moieties attached thereto, e.g.
conjugated.
The total number of spots on the substrate will vary depending on the number
of
different polynucleotide probes one wishes to display on the surface, as well
as the number
of control spots, calibrating spots and the like, as may be desired depending
on the particular
application in which the subject arrays are to be employed. Generally, the
pattern present on
the surface of the array will comprise at least about 10 distinct spots,
usually at least about
20 distinct spots, and more usually at least about 50 distinct spots, where
the number of spots
may be as high as 10,000 or higher, but will usually not exceed about 5,000
distinct spots,
and more usually will not exceed about 3,000 distinct spots. In many
embodiments, it is
preferable to have each distinct probe composition presented in duplicate,
i.e. so that there
are two spots for each distinct polynucleotide probe composition of the array.
In certain
embodiments, the number of spots will range from about 200 to 600.
The amount of polynucleotide present in each spot will be sufficient to
provide for
adequate hybridization and detection of target nucleic acid during the assay
in which the
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array is employed. Generally, the amount of polynucleotide in each spot will
be at least
about 0.1 ng, usually at least about 0.5 ng and more usually at least about 1
ng, where the
amount may be as high as 1000 ng or higher, but will usually not exceed about
20 ng and
more usually will not exceed about 10 ng. The copy number of each
polynucleotide in a spot
will be sufficient to provide enough hybridization sites for target molecule
to yield a
detectable signal, and will generally range from about 0.01 fmol to 50 fmol,
usually from
about 0.05 fmol to 20 fmol and more usually from about 0.1 fmol to 5 fmol.
Where the spot
has an overall circular dimension, the diameter of the spot will generally
range from about
to 5,000 Vim, usually from about 20 to 2,000 um and more usually from about 50
to 1000
10 um.
A critical feature of the subject arrays is that at least a portion, usually
the majority,
of the polynucleotide spots on the array are made up of polynucleotide probes
that all
correspond to the same kind or kind of gene, i.e. genes that all share some
common
characteristic or can be grouped together based on some common feature, such
as species of
origin, tissue or cell of origin, functional role, disease association, etc.
Other spots which
may be present in the pattern include spots comprising genomic DNA,
housekeeping genes,
negative and positive control genes, and the like. These latter types of spots
comprise
polynucleotides that are not "unique" as that term is defined and used herein,
i.e. they are
"common." In other words, they are calibrating or control genes whose function
is not to tell
whether a particular "key" gene of interest is expressed, but rather to
provide other useful
information, such as background or basal level of expression, and the like.
The percentage
of spots which are made of unique polynucleotides that correspond to the same
type of gene
is generally at least about 30 number %, and usually at least about 60 number
% and more
usually at least about 80 number %. Therefore, the arrays of the subject
invention will be of a
specific array type, where representative array types include: human arrays,
mouse arrays,
cancer arrays, apoptosis arrays, human stress arrays, oncogene and tumor
suppressor arrays,
cell-cell interaction arrays, cytokine and cytokine receptor arrays, rat
arrays, blood arrays,
mouse stress arrays, neuroarrays, and the tike, where some of these
representative arrays are
described in greater detail below.
With respect to the polynucleotide probes that correspond to a particular type
or kind
of gene, type or kind can refer to a plurality of different characterizing
features, where such
features include: species specific genes, where specific species of interest
include eukaryotic
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species, such as mice, rats, rabbits, pigs, primates, humans, etc.; function
specific genes,
where such genes include oncogenes, apoptosis genes, cytokines, receptors,
protein kinases,
etc.; genes specific for or involved in a particular biological process, such
as apoptosis,
differentiation, cell cycle regulation, cancer, aging, proliferation, etc.;
location specific
genes, where locations include organ, such as heart, liver, prostate, lung
etc., tissue, such as
nerve, muscle, connective, etc., cellular, such as axonal, lymphocytic, etc,
or subcellular
locations, e.g. nucleus, endoplasmic reticulum, Golgi complex, endosome,
lyosome,
peroxisome, mitochondria, cytoplasm, cytoskeleton, plasma membrane,
extracellular space;
specific genes that change expression level over time, e.g. genes that are
expressed at
different levels during the progression of a disease condition, such as
prostate genes which
are induced or repressed during the progression of prostate cancer.
The average length of the polynucleotides on the array is chosen to be of
sufficient
length to provide a strong and reproducible signal, as well as tight and
robust hybridization.
As such, the average length of the polynucleotides of the array will typically
range from
about 120 to 1000 nt and usually from about 120 to 800 nt, where in many
embodiments, the
average length ranges from about 200 to 700 nt, and usually 200 to 600 nt. The
length of
each polynucleotide on the array is less than the length of the mRNA to which
it
corresponds. As such, the polynucleotide represents only a fraction of the
full length cDNA
to which it corresponds.
As mentioned above, the subject arrays typically comprise one or more
additional
spots of polynucleotides which do not correspond to the array type, i.e. the
type or kind of
gene represented on the array. In other words, the array may comprise one or
more spots that
are made of non "unique" polynucleotides, i.e common polynucleotides. For
example, spots
comprising genomic DNA may be provided in the array, where such spots may
serve as
orientation marks. Spots comprising plasmid and bacteriophage genes, genes
from the same
or another species which are not expressed and do not cross hybridize with the
cDNA target,
and the like, may be present and serve as negative controls. In addition,
spots comprising
housekeeping genes and other control genes from the same or another species
may be
present, which spots serve in the normalization of mRNA abundance and
standardization of
hybridization signal intensity in the sample assayed with the array.
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Polynucleotide Probes of the Arruys
Each spot of the pattern present on the surface of the substrate is made up of
a unique
poiynucleotide probe composition. Iiy "polynucleotide probe composition" is
meant a
collection or population of single stranded polynucleotides capable of
participating in a
hybridization event under appropriate hybridization conditions, where each of
the individual
polynucleotides may be the same -- have the same nucleotide sequence-- or
different
sequences, for example the probe composition may consist of 2 different single
stranded
polynucleotides that are complementary to each other (i.e. the two different
polynucleotides
in the spot are complementary but physically separated so as to be single
stranded, i.e. not
hybridized to each other). In many embodiments, the probe compositions will
comprise two
complementary, single stranded polynucleotides.
In those polynucleotide probe compositions having unique polynucleotides, the
sequence of the polynucleotides are chosen in view of the type and the
intended use of the
array on which they are present. The unique polynucleotides are chosen so that
each distinct
unique polynucleotide does not cross-hybridize with any other distinct unique
polynucleotide
on the array, i.e. the polynucleotide of any other polynucleotide probe
composition that
corresponds to a different gene falling within the broad category or type of
genes represented
on the array. As such, the nucleotide sequence of each unique polynucleotide
of a probe
composition will have less than 90% homology, usually less than 85 % homology,
and more
usually less than 80% homotogy with any other different polynucleotide of a
probe
composition of the array, where homology is determined by sequence analysis
comparison
using the FASTA program using default settings. The sequence of unique
polynucleotides in
the probe compositions are not conserved sequences found in a number of
different genes (at
least two), where a conserved sequence is defined as a stretch of from about
40 to 200
nucleotides which have at least about 90% sequence identity, where sequence
identity is
measured as above. The polynucleotide will generally be a deoxyribonucleic
acid having a
length of from about 120 to 1000, usually from 120 to 700 nt, and more usually
200 to 600
nt. The polynucleotide will not cross-hybridize with any other polynucleotide
on the array
under standard hybridization conditions. Again, the length of the
polynucleotide will be
shorter than the mRNA to which it corresponds.
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ftrruy Prepcrrucion
The subject arrays can be prepared using any convenient means. One means of
preparing the subject arrays is to first synthesize the polynucleotides for
each spot and then
deposit the polynucleotides as a spot on the support surface. The
polynucleotides may be
prepared using any convenient methodology, such as automated solid phase
synthesis
protocols. preparative PCR and like, where preparative PCR or enzymatic
synthesis is
preferred in view of the length and the large number of polynucleotides that
must be
generated for each array.
For preparative PCR, primers flanking either side of the portion of the gene
of
interest will be employed to produce amplified copy numbers of the portion of
interest.
Methods of performing preparative PCR are well known in the art, as summarized
in PCR,
Essential Techniques (Ed. J.F. Burke, John Wiley & Sons)(1996). Alternatively,
if a gene
fragment of interest is cloned into a vector, vector primers can be used to
amplify the gene
fragment of interest to produce the polynucleotide.
1 ~ In determining the portion of the gene to be amplified and subsequently
placed on the
array, regions of the gene having a sequence unique to that gene should
preferably be
amplified. Different methods may be employed to choose the specific region of
the gene to
be amplified. Thus, one can use a random approach based on availability of a
gene of
interest. However, instead of using a random approach which is based on
availability of a
gene of interest, a rational design approach may also be employed to choose
the optimal
sequence for the hybridization array. Preferably, the region of the gene that
is selected and
amplified is chosen based on the following criteria. First, the sequence that
is chosen should
yield a polynucleotide that does not cross-hybridize with any other
polynucleotide that is
present on the array. Second, the sequence should be chosen such that the
polynucleotide has
a low probability of cross-hybridizing with a polynucleotide having a
nucleotide sequence
found in any other gene, whether or not the gene is to be represented on the
array from the
same species of origin, e.g. for a human array, the sequence will not be
homologous to any
other human genes. As such, sequences that are avoided include those found in:
highly
expressed gene products, structural RNAs, repeated sequences found in the
sample to be
tested with the array and sequences found in vectors. A further consideration
is to select
sequences which provide for minimal or no secondary structure, structure which
allows for
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optimal hybridization but low non-specific binding, equal or similar thermal
stabilities, and
optimal hybridization characteristics.
The prepared polynucleotides may be spotted on the support using any
convenient
methodology, including manual techniques, e.g. by micro pipette, ink jet,
pins, etc., and
automated protocols. Of particular interest is the use of an automated
spotting device, such
as the Beckman Biomek 2000 (Beckman Instruments). As mentioned above, the
polynucleotide probe compositions that are spotted onto the array surface are
made up of
single stranded polynucleotides, where all the polynucleotides may be
identical to each other
or a population of complementary polynucleotides may be present in each spot.
SPECIFIC ARRAY TYPES OF THE SUBJECT INVENTION
A variety of specific array types are also provided by the subject invention.
As
discussed above, array type refers to the nature of the polynucleotide probes
present on the
array and the types of genes to which the probes correspond. These array types
include:
human array; mouse array; cancer array, apoptosis array, human stress array,
oncogene and
tumor suppressor arrray, cell-cell interaction array, and cytokine and
cytokine receptor array,
as well as other types of arrays, e.g. rat array, rat stress array, blood
array, mouse stress
array, and nueroarray. Each of these arrays is described separately below.
Human Array
One specific array type provided by the subject invention is the human array.
In the
human array of the subject invention, the majority of the spots on the array
have a
polynucleotide sequence corresponding to a human gene of interest. As such,
all of the
unique polynucleotide probes on the array correspond to human genes. The human
genes
represented on the human array are typically those genes that have been
identified by those
of skill in the art as key genes. By "key" is meant that the genes are
relevant and related to
the purpose of the array, e.g. the identification of difference in the
expression profiles of
different cell or tissue types, where the key genes are generally functionally
important to the
cell. In many embodiments, the genes represented on the human array are
tightly regulated
human genes. The term "tightly regulated gene" is used herein in accordance
with its art
accepted definition and use. As such, by tightly regulated human gene is meant
a gene which
-13-
CA 02290738 1999-11-19
WO 98/53103 PCT/US98/10561
is not "leaky," as opposed to housekeeping genes which are generally expressed
at similar
levels in different cells and different tissues, i.e. a gene which is
inducible such that in
response to a specific inducing signal the gene turns ''on" and when this
signal is removed,
the gene turns "off."
In certain embodiments of the human array, human genes that may be represented
on
the subject arrays include: (a) oncogenes & tumor suppressors; (b) cell cycle
regulators; (c)
stress response proteins; (d) ion channel & transport proteins; (e)
intracellular signal
transduction modulators and effectors; (f) apoptosis-related proteins; (g) DNA
synthesis,
repair and recombination proteins; (h) transcription factors & general DNA
binding proteins;
(i) growth factor & chemokine receptors; (j) interleukin & interferon
receptors; (k) hormone
receptors; (1) neurotransmitter receptors; (m) cell surface antigens & cell
adhesion proteins;
(n) growth factors, cytokines and chemokines; (o) interleukins & interferons;
(p) hormones;
(q) extracellular matrix proteins; (r) cytoskeleton & motility proteins; (s)
RNA processing &
turnover proteins; (t) post-translational modification, trafficking &
targeting proteins; (u)
protein turnover; and (v) metabolic pathway proteins.
In view of the length of the polynucleotides of the probe compositions of the
spots,
each polynucleotide of a probe composition typically has a nucleotide sequence
of only a
portion of the human gene. Specific sequences to which the polynucleotide
sequence may
correspond include those identified in Table 1 below, where by "correspond" is
meant that
the polynucleotide could have the same sequence as specified or a sequence
complementary
to the specified sequence. Whether the polynucleotide sequence is the same as
a portion of
the sense strand of the gene to which is corresponds or complementary thereto
is based
primarily on the nature of the target which the array is to be used, e.g. if
the target is first
strand cDNA, the polynucleotide will have a sequence found in the anti-sense
DNA strand of
the gene to which it corresponds.
Of particular interest is a human array of the subject invention as shown in
Fig. 1. In
the array, each spot on the array comprises a known polynucleotide, as
specified in Table 1,
where the array comprises spots which: (a) correspond to 588 different tightly
regulated
human ~~enes; (b) comprise plasmid and bacteriophage polynucleotides; (c)
comprise
polynucleotides corresponding to housekeeping genes; and (d) genomic DNA. Each
of the
different types of polynucleotide spots are positioned at a known location on
the membrane
surface.
-14-
CA 02290738 1999-11-19
WU 98153103 PCT/US98/10561
TABLET
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O N O (O~t MN COO M N~ ON .~00n '~r N
0 'VCOOM
r r ~IwO _ ODT (pOM (DI17O C~dDO I~O(DO 1~Inf~~1 ~f7raD~ 7r 1nO OIn
C'~ f~M ~ 00ODQ'M Il)N InMN CDMr r Or C~OO V Mtf7V'NM MO
r M rr r r MM cf'MN N MO r ~tr ~ MN r (DM O NM M r(~OpNO N r (pr
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lt1
-IS-
CA 02290738 1999-11-19
WO 98/53103 PCT/US98/10561
TABLE 1 (CONT)
i
~O ~ CO '~' t0 CD ~!' O N f~N M N MM M NCDN In r
C M'O' VCDi~ NC'O t0~t O V'~I~tnOO M O ~ h.c0N M I~M O O M
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N (~ r _'~ T T r ~ ~~ ~ r' ((~ r
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!fCON 07 ' ~ MN 'a'Cf~O ODO N DOh ' '
(D
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lf~(fl O CON M ~~tf~~fr InM GOr N InN 1~d'
Q.~'N d'Mf0N tnlf7N O (Dr (Dr(pr l1]ODr CON M NN N Nr O r rM 'v100r tnr
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d1Oo ~nNl~,nI~~CJU CC~C~C~C~C~u7C7C~pC~C~CJC~C7UC~C~C C9U C UC~IC~C~
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-16-
CA 02290738 1999-11-19
WO 98/53103 PCT/US98/10561
TABLE l (CONT)
I
f
O ~ r O N M In O M O M O I'- 00 M (O O O Lt7 N V''r pp LI~ I LO
M C' r CO O 117 f~~ O CO N 40 N Ln ~ Li~ M I~ O O h- d0 M ~ QD r M ~ 1 O (O
N O r O CO I~ M O ~ r O CO ll~ O ( O 1~~ M O f~ In f~ ~t M In CO C (D M c'~ OD
V ~ ~ N O
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M In (D (v r ~ 'ct (D M CO (O r ~ '- 00 O M f~ V' M M r f~ O ~ ~ ~_ '- r O M M
~ ~ I r O
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1~ 1~
00 OD lf7 (D ~' O C~ N (O (D In tn CD 00 V r <D M (O (D CD ~' O O In V V r O O
CD O O ~ O In
(1 r r N r r M M N r r r N M N r M r N r (p O 00 r N CD r N f~ f~ r N r N r dD
M
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t~ v o n. ~ c a H a a~ v a~ ~ U a a v ~ ~ °
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t0 N ~ c0 O O N r M N M d' t~ N O 00 CD M M O CO CD t~ N cD O~ O) ~ O
m h O O Ln ~ 00 Q) N 1~ r (O O 00 r ~ OD (D M N ~ (D O r ~t V' tn O M (D ~ i'~
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d M O O ~- ~ V ~f 1~ h CO CO OD r M N r (D 00 ~ ~ ~ In tn tn h. Q7 O r N N Ln
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an~u m~c~~o~mmnaiomlommlunuumo~u o~aa~ooonimamoa~i.o~o~a
_ 17_
CA 02290738 1999-11-19
WO 98/53103 PCT/US98/10561
TABLE 1 (CONT)
t I
00M MODI~Mr N (D N n 00l l7COO N N OOM M
M r 00GD~ rM ~ N I t1' r 00 00CGOO I~ O7f~r CD Inr 00MOD
r N M l l~ t
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~ ~ 1~ N(
M M N NM r Nr N rM r r MO NN r rM ODQ'Nd0N (Dr ~ rN M rr lI700I~
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1 O T'I~M f~O ~fO0 0 0 ~I~Mr 0 ~~ C0(000'~O ~N N ~MO ~ ~r ~ ~N
OO O ~ (DO d'tnM(OCDCOCOf~CO00N N tI7CO ~N
N C~C~C~~N M MO (~OD ILL_ DD MO ~ YJ ~ J ~J J J_ J _IJ ~ rN N N~
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-18-
CA 02290738 1999-11-19
WO 98/53103 PCT/US98/1U561
TABLE I (CONT)
-.
_ -~.N - ~
r O O O O _
C ~' r ~ ~ ~~ ~ ~ ~ I~~ r O d0O N MV O CN~7VN'
I I
O fwrC V'InrIwr ~N 1~Nr N fI O d0CON r(O(D O - 1~'~'O N r~ N NM N
O O r M CO00N O O -O (DrN CON Ln f~
'vtI I
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~ ~ l0I~m M lI~07 ~ M M M ~N
1~~M M h Mr CpOaDO M~ InN~ N lf7COO ~Y07N InInM 00NO (O1~N 'C'Inlf7
O (p~~ (p~ Mr fvNO 00rf~f~OO M VO O Inr OD~-N r M NO O r00COOr O
O.tnrN M f~d0In'~'Mr (Dfwr r (pN r rapr r~ O (DfwLI~M (OCON rN r NM t1'
r
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N
m COO~ ~ fDCOO Q (O1 - rN M OO I~rO r00r MInO r NN ~ O'~Tr rtI)M
Cl~ C'V'InCONh r r~ ~f1~I~N MtnI~00O)N 1~O r NN M InInInf~dOM V (OM N
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a m lt~u.Iu.Iliu.u.I~mu.Q ti.Iim l~tillimt~;u.~tiu.~m lm~ tiItLU mIwt,L~~~:'~t
QIm.~
- I 9-
CA 02290738 1999-11-19
WO 98/53103 YCT/US98/10561
TABLE I (CONT)
I
v
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c1~ r(DO (Od0 I r O M I n ~ M M O f~'~ '
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O r r rr r lnN r r r NO r NQ7I rtr O ~r LI)NODr rr O rN O I l7
~ 1
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r 1 (DM r (Dfs COC~nn Q7 r r ~~ tn~r cDIn D
O O Wit'O 00 r M ~ N M 1~ ~ I IM O 1
ttN D 7N I~ON GOIN O
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p ~ O N1~N N~ O ~ M O!~tnOM M rOpI~Iw N 00~ D r M
( ( In
N.r tnrr r rN r r r NN h NM M rN M rM tnrr 00rr r r00r N M
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C N O Op~r ~N M C~ M cr r rr r ~0 0 00 0 rr r lnl171nlnln(Dl~tl~l~
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.t ..I tl .t U .. .u l. .. .... t
i. l
-'7 ~-
CA 02290738 1999-11-19
WO 98/53103 PCT/US98/1O5b1
TABLE [ (CONT)
I
I
I
rInN Of~ O~ ~ V 00f~r O M N ~th-r ll7 r 'ctI~~ O h
I 1~ 00 a1 O L17InI O M O lf7 M CD M
r 00
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GOh InInti7 (D~ 1f7O f~00Nr ~ rtnCOt~.Nr r InN ~1'Ct17COd0f~N (D r (p
f\rr r rN M NM N 00NO ~M r rr r M ON N OCDr h1ON rO 00t0r ~ M
I I N
CO r r M M ' 07'~ ' 00 r M ~-r1~. (Dtn rr ~ tn' Is
O N M ~
N COIn( 1~M ~ r~'M (~f0O r~ (OCDN ~ M COIAN 0000r tnf~f~d0ODO NI O CO
D ' - OM r InO r tnr N C7O 7
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a O9dtoO CON M t1N r O M~ OCOr ODN V OD~I C l~~ r Mr N f~COCDtOCDIr M
O rO r ppN GONr N I~NcIMN r Mr r O r~ dO
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O OM M Ot7M M Intnf~r (OCO(DCOODODM M M V''~~f77I~COr! ('~MI ! V'CO r
I I~ r M V I I
M O O
N O rr r rr r rr r N NN NN N NO O O OO O OO O ~ rlr rr~r
C9>-QO D IDD D DO O D DD DD D Q~ ~ -~-~J J JJ J JJ r ,JIJIJIJJ;J IJ
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Q ~t~im II.uQ mim
_7 [ _
CA 02290738 1999-11-19
WU 98/53103 PCT/US98/1OS61
TABLE 1 (CONT)
I I I
i I
i
i '
O OI O O M O O M r O COr 0000
C Op(D 00rtf7r N M00O NN M OCDIn~LOCON~ 1~f~O V''V'r tn r N rO M
O M rt!7N t~M N Q'r~ ppNN I~NN In1nr N rO fDOr r ~~ O O 07N ~ NM
~
~ VO ~ ~lt7I~r ('~N 1~rI~tnf~r f~Mr f~r(D(Drr r 00Inrtr (D(OC~~~'(D
~ n 00 n M NN N
M I~M M MI~I~CO1~I~O O(DItjInO ~ LnN N V'r I OI~t 00 I ~ InO
~Or MO InCDN COCOON d0(DO Inr1~InrN (ON(OCOV(O00MODtnO CON ~ f~CEOr
d r raD' rM r M rN VIC)~fM d'O ~ rI N 00~fM 1~I~ m ~N N tt7lnM d'rN M
N rI
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. d
c OC C O O ~m ~ '~V p~ -T M t0M <fCDNM
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(D(OO r NM LI71hl17V "cTMO)r r(O~'I~M ~ MO M OM O Or r r rr r OpOD~V
O ~~ N NM r ~ h~ (~lI7Inr 00~ (DCepM N N NN N ODlMr
N O
COOM M IC'COO OC tnON lf7ODO N tnN r O~ ~ Or r ~N 00OO00CADCO00m O
C ~' ' ' n M ~~ ~prI M COOD
N
O OO O OO O O NN V VInO rN M ~l I ~rr NONN NN N N NN N jINN
d ~rNN N NN N N NINN NN N MM M MM ~ N
C~J JJ IJJJ J J ~JIJJ JJ J JJ J J~JI~ ~ ~~~I~~ ~~ ~ ~ ~~ ~ I~I~
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~. IU~mm mD ll~m ImN IUmlmU Dm d Om d OlmO wm w dm m m tmlmm dI
M w IU in
Q~m lUld
_77_
CA 02290738 1999-11-19
WO 98/53103 PCT/US98/10561
TABLE 1 (CONT)
iI
I
C O O ~r-tn (Det 01 O r r.~t00f~N c0O d0InCDM f~ NM C
I V (DM ~D (Ot~ M O O ~-M ~M OD O ~ t0
In ' tn '
rJ'117I~(DtD(D'~m O O Mtf~O N r 00N ~ NO Cdh-V ODON d M ~O LOd7O Q9
' ' ' 1 ~
r N NN NM N r ~N CO~fr COODC N rN r O( (ONr r CDM M M rO rO CDM
(O' tnr h 00 1~M07r r r~ (p'- ' ' V' O r
CO00OInf~ O O M (D M '-CO cD~ CO00OInf~ tn O ~ CO
' ' i~ M O ~ O ODM O InOO O(D f~
InO VCOI(DO N N M ~N M O M N N O r 1 .
O O MN ~r r IW'~~'M (DN ~ 00~'ll~OtDr ~'(p~'OpO '~(DO r Ice-NO NO O (D
N
Q.r N NN NM N r tnO ~ Inr ~ ll~COO 00~ e-~M (DOr r LON M r e-r.r~ ~'M
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InV Mr MM M f01~(Dlf7(OODN MN rO ~trO r-NM 00r~ ODN f~r In~ (ON
N ~f'
d N COON Itf7COdOCDMO M OO ~t~tnh O0 0 0M ODCDC~O'~Y'cDaOM tnr.C010tnI~CD
C O O O~rrr r N MQ'V "~(D(OfD~ tO~~'O ON N NN M C~M V ~f~t~ltnCO(DO
d M M MM MM M M C~M M MM M M<DM ~tnltntOCOCOIO<DCOCDCOI~.t~I~11~1~f~l~CD
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a~w O,p
-23-
CA 02290738 1999-11-19
WO 98/53103 PCT/US98/10561
TABLE 1 {CONT)
I I
I
Oln N rtD'O Mlnr ~ Q7N ~'r(D MN M M IM O
~ OD~n D N ~ r d0l)CO~N n N COO D DD C'ODl~ MN M OD~V' ~t
IC ' l I ~I ~( C C l f I'
~
C M Md0n MM 00 ll)M ~ r~ D ~N ~1'M M r~ n MO ~ DO COMn O M Or- r
O MI~I r Or NII~r-1 N f( i NN r rO r fI NIn' ( Ir I Ir N N NN N
O r rr O NIr N N r M r
lncDl~'r r-r r M~ ~'r r~ r ~r ~ C'O ~ NO r MO 070DN ~ N(ON ODrO CO
~ ~M N ~ - N 00O r V'N M 00 hInM M NM MII~rII~~OM O O O~ N
O f~~M (OO~fCOa0O ~ 07Nn O ~C)O N00O 00V N OD O MN N ON f~O Mf~ CO
N r d'~ ( I (
N lf7r Q7~(O4DO7r r 00~r ~ Nr r r~ r rr N nr N rr r rr r N ~r r
( I 1 I
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O r Ot~O ~O OOIM M M IfsM V ~I~~OInO r NCOaDMM (Od'QOh O( LOM (DCO I~
M 'O O
Q>O Int!7C MN M MO M ~ttf~ODN tl'CAD~ ~(OdO00N Cflrd0InmOD~h~V 1~M Mr 1~
(plwf 00Na0M MtnN dONV'aDO)N (OOr O OM M (OCOO ~CDOOOM COr N(D
r ry r Ittf~~h N ~ Mt!7(O(O~ f~~r N NN ~ NN M MM ~ ~~ ~ I~f~I~ n
ppppr apMODO~1~a0O~N O~O~O~O~I~m (DO O OO N OO O OO O O~ O O OO 10
pp a0 00 O~ p7 O O
p~
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E Ntn~ ltdN (Dt l17inLna>InInt In~tff7~ .cInLnInInN tn~!' C~tf7'Vr ~co
~ ~aIao oU IU~ o o IO~tm oIno unam 'o o oo a om (Ooa lmm mI
Q~o (D u ,o ~ ~o CO
In lm !alm
iUC~
CA 02290738 1999-11-19
WO 98/53103 PCT/US98/10561
TABLE I (CONT)
i
O '~'O00 M'N (D Lf7 ~ M 00MM N N fD~'MIn r lflM 1~ O
L.M 1~-N( ~DO O ~' V CON O I O~ (pI V'InODV M GOr M 00 N f\
N r tn ' I
I
r M O~ M O00COrN (Or(pN O'"'Tr OM (O(DCDN MN r CpO V r ~IwO OO I~
M N ~r M NM M ChO N NN C'tl~r N ~V'N N NN rN r InO N M NN (DId N
ct
lfy~ htn~ In(Dr r~ M ~CO~ ~O (OrN ~ ~ ODl0f~O r 00r apO M~ ~ lf7~
'~
Q M M L(7Iw1~V't17f~ON f~r~'InQ)~ r NO InN M(OOI~~ M47M ~-NM Q7d'
I~O (DtI700f~In(DO)M N InN (DI~O 00f~O M N MCOO~ ~ d'M r GOrN COI~O In
Q.N N V'r Q rM f~Nr N ODN CO~fr'r M~ N N Nr rr ~ lL7(ON N Nr N MN N
I
N
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7
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(S1
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'
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l7 o '
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r r Otn(DI~O N NN M NO I~Inh M d'N r r ld'M NO M IfIN I~r rN M M
ODO COInII~(OM MV'1nO00U7~0~0 [vO N N 00COrlI7IwOM M M ~O N ~~00
C 1~I 00COO OO O OO N MM ~tcttn(OCp1~Inlf7(Dr lf7InO 1~O Q'tDOCOO 00~
00 I
d O IOOO O OO r e-r r rr r rr r OO r r r~ 6ntnInCDf~I~N OO r NO r
('O ~~>> _ >> > >~>> >O > >~ O ~XX X X IXX XX X XX X N QQ O O-~J
d
r
ca
~ I
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I
U I I
~ U f'O47E ..-C QfL.._ ~C EG ~ G c0.OtU NO ~ ~~ U U~ N ,
~ COIf0~fcDr1c0COfDt0NcDcDcDCOf CON M M c0M I~c01~U1nInCO1~1~t~.V'
~ IOtDO lmOm O O~O;OIUO O IOO tnIUiQQ Q u~Q OQ IO(~m m w u.w ~ 1~
w IO Im IO I
cD ~
Qiw w~~lu.I
-25-
CA 02290738 1999-11-19
WO 98/53103 PCT/US98/10561
TABLE 1 (CONT)
I
M r ~IN l!7~ O 00 (DM O) N ~ ~t In f~
~ ~ M NO 001~ COO CD N COI~r OD I 400)O
I
M CON ~'M M M '~~ r O)V'Inr!~ODOaDtnQ)In(D1~ 40COr M N dD(DNIn(DCO
001~I~Ind'(O~ N r~-N rO)lf)NN C ctr tnrO M MO M Or ODV'rr I~; r f'
~- N r N V
' I
r tn' I~~ r r CD00r l (7 V' ' N ~ Q)Inll~N ~-~ Inf~~ (D
N (D O ~ M ~ N COQ).O Vr O
OpNr N r07O)d0OM t~M1~M (DODU7rN N OQ)~tN~ ~7~M I~M Mll7OV'CON
N I~~'N ln00l17M Olnt~(DIM (D(~M ~f~ttnl~l~Ir N ~fN N OO lf70)mM O7tnM r
a 00(DN r V''~Tr Q)~ODr rInInCON N N~ r r1~M Mf~COInN N M aDr NV'r'~T
If 1 I
I
i C_
.p
i
m
I C T
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a L p U ~d
p~ N
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m O L ,: N C
a ~ a ~~ ~ ~ ~~m
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I
c ~~ o ~ c'aU p U ~~ o
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n
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O . d v. . . + N
dd dU N 'O d ~ O p N ~p N 7.~
Op, ~O p N C~ ~ .C C cCQ O 7. ~np vn v~ "~ ~
'.' U
m C.-o a>_ cna~ .a ~_Z~- _-- cn~ c'c'am r a~~ n
U Od L Q ~ ~ - d Q
O ~ a i'
.Cp YOd U Y p_CC ~ Q p ~ L. N ~ O Od ~ cLf~ .
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c~ a om ._ ~n~ a Z ~ncna~'ca ~~ ~ N -c~m~n~n a~ ~ ~ o
. ~
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C 4) C~ ~ ~ 'D~ ~ E E C C C a I
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~ Z7E.D~ N N. N C ~ c ~N ~U ~ N '~NN N OO O I
E ' _n O O
~ ~ p~O U d a 0 ~ O O .~ UC U~_C ~ CC C UV C U
C~ U l .
~CI U Q ~C~C ~ 'j~ ~ QO O _~ ~'..,O~ O ~s LL L O OO s O O
p Oi
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U
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d 0. d -~ U -U C UZ Z OZ O OC O'U,pZ _7_7_~U OOOI C O
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r 0~ (OMI~CDr MN N Q)~ O MI~~ ~ln(OM(DQO~tO)~ ~t~ COODQ)CO~tnIMQ)
COO(DCD~cO~ ~t~D~y CO O Of~N
~ 01Nr O ra0V'00npp - N M (DfD00rr ~ ~V'~ InI ODOO r-r N1~d0I I
C ('~~!f~O)r00(D(D(pppO OO M OI~I~~-r r rr r rI~r NN N N NN Na0O M
d r rr N McDI~COOlOI f-H-H ~I-f-Hf-f-I-I-f-I-I f-H~'-~-I--I'-I-I-'N M E-'
I- ~ r 'F-I
I F-
H
C3~ ~~ ~ ~~~ ~ ~ ~X Z =Z = Z2 Z Z2 Z Z= _ __ ,=Z= Z Z =2 =.2.=12
N
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( C U Z7
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Q ~ tl~'~ I~I~~ luWf~IQIQQ U ~aU U UIUIQIQ~UQ QIUIUUU IUQ QQ ~Q; U
It!l~Iu..tim Lu. C
IU
Q
_y_
CA 02290738 1999-11-19
WO 98/53103 PCT/US98/10561
~1'ABLE I (CONT)
I ~
I I
I !
I
1 ~
O r N M M 0M Q' r ~ 1~ N 0~ D (D NN r M
' '~ I (D ~'~ d NO D O V'cD 0 I( ~ ~ 40~ O M V' O
1 fa ~ (( G O aDm I ~ '
f O~
C DN u7QN LI) NN V rM r nO CpOO)O C'~V OD O r0 O ~N O O DO r
O O D OOD ~ N I NMIN Mr NlrN N (f7N aV 1 ~C C ( MI C'7
C C C ~ l O d' N N N tD
Nlr
d0N C'J~10 CD MO C1r00h ~Oi~ OO N N~'CO Mn O ~tD ~ 0C0(D r ~rIr OD
Wl ~r OOC O'- r 0D N
N GO7 N OO N n~ h OM 00~O (OCOr D O r ~n O f~N D O~ O N Nl7 00
(D ~ Wit' t I ~ f ~ ( n ODO ( tM l LnC r ICOM n lO ~
f C I D( D l17 ~J' t 00N
I
O
1
O O~ M ~OU00 nr r f7O 00N. M DD N CON r Mtl7N N~'~ rr N O VMIi~ N
d 1rI1 MI~ I Mf~(l ODN N fN t (r N
r M tn O N N N
I i
M r,
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0 0 0 '~~ ~ ~ a c .~Wm ~ 'E ~ ~cn~ Y ~ M v
a a ~ c N ~~ c n~ o '~ c o0 0 ~ co ~ z
c .o . co ' a.?~ m
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U
E U V~~ E E OE O ~~ ~ ~ M ~47N c0 tn p p~ C C
~ '.'~'Q7 ~ ~ N O Op Y .Y
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d M O sa cb" ~ Q'~ ~ _ -p O C I
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~~ _oaE E E c '~~'-:~~Icoa~o~a0m a~._- ~~ ccmo c oa Y ~ co c G
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~ p ~ U .
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U a a~ "_
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I O I
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~
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COt~O MI O f~O h 00M ll7MN ~ OCOr In(pO Cp1~N In(DN 1~M N GADrM , O
~ O
rM (flr O V''ctV'(DO ~ MO O OO r O~ I~00CO~hOO T ~M M r- lf~1 f~
M ~r O~(ON Q7Inll7r'N1~" O M NN r CO
I
I~
N NM V tnCON N~ ~ rN _ ~N ~ ~~ O rfDI~MM CO~'O O Or
C MM M MM d' ~tV 'C'1n .a. r MM ll~000 0 1171n(DI~O M vYM I~COI~tnl(DCO
o
N f-F-I~(--f--1- I--1-I-E-O O p,-~ I~'-~ I~X X XX X X7-D D~ l J JJ ~JJ
~
C9== Z =I=Z ZS Z Z~ '~-~-1 2 , ._,
C_
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O
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OU ._~C~..-'D N._. L.~Y -E cb~ U 'ON .~Of .__..Y 'O~ O y I
M 1~ I~O M(p(Of0C ht~1~1~I~-~ f~.Ch 1~ M r~ v DIIL
O (O t~ I~- W vnco
M (D l l i
~c7
mn
a.1~f~-c0M~ U U( Uw uJu.mnu~ umuu.fm >.~ww ~ ww U lmm lmm ialmlmlmi
Q U,Ua alU alU u
CA 02290738 1999-11-19
WO 98/53103 PCT/US98/10561
TABLE L (CONT)
i
I ca
r (p(DOO r (O ~t COCO N I~~ O N M I (D I M
I
C ppO [Wp r r N O ~'7 ~ N07N CO M 00 Lf7O ll7~fLnd' N
ODr (O(Or Cpr COInO O CON fDMN lf7N Q)O N rCpNO r M tL7C'CpI~O M I~dpM
O r M r~ (pInM r rM M 00CDr fDr r ODrapr ON ~M O Nr O f~'cf(DOr CO
~ '
arr 00r ' 00 ODCO r ' Cp [~r O O r r r In1~(OOD CC
~ InC~f~ r In 00 n tf7N r aD O 00~ tn r n
n M NO V rO (DOM fDCO~'O OM ~ (D00O COC~ OInO InOO 00OO COI~r M
r I
(DCOMM O Nt~tWMM f~N~i'N Q)t17N 01~aDV NN In(DO ll7CDN O 00CDI~ c0N
I
C.1~N Tr tnll~O r rLnN InV r [Wp r M r~'COInN 1~M r 1 Q)r 1 M~ M (Dr
M
T
N
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c U
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ih
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C tn ~cA0 0, ~ ~'C v~ ~ 0 ~~ .=(O ~~ ( r N pU I O
~ Y O
rE Ll~m -00 ~~ ~ i ~ o or (~N CS U ~ ~Q t~ t
E ~~ c ~ ~ ap a~>a ._-o~ ~'d
I
C N v oc . d . _a c ~O ~ a ~ ~ ,Q YO c0c cc cn~~ N rC
Q- ~ ~ 3 ' U T - W p .
~ >'E ~L E ~~ T ai X N Ma ~ ~z ~~ ~ j,~,>. OQ ~ mN O
Q Q D.d > I
v U- J ~U Q UU N.Q7U U Q= O CnCnC~U N.~ UU ~ U UU Z LU ......mO L
.
i
i
C
00O OCOO NM Lf700I M ~(ON GDr O O rp ~ M~ MN COM ~ODr Of~(D1r O
~f~ I~
N O OM N N~ '~tLn(DO 1~(rJr ppN ODO a7p p ~M ~N d'r rM InfWO 0 0N 1~
C1N V'OO f~1~1~~ rCDN OO faON ~tr r~ Q ytCOMll71~~f~GOf~Nt~I~Itf7O
I~
C M Intn(OO OIn1~r'~ ll)OInM ~M ~'1~!~p cl~OInOM M f~~(ON I~(~fD(Dr C
y r r ~r;rr ~N N MM M CD(OI~It~dO00~ O~ N MM ~~ ~ ~f~tCDO 00 r '~7
C'3~ W ~~~~ ~~ ~ ~~ ~ ~~ I~~~~ ~ ~ ~f~~ ~> >~ ~ ~ >> X IXX X XX X
U
..-E E O C ...~O ~C41L C_ U c0~U ~ ~C.ON .r~ -
E
c''ic~cinc~vcain~iWn mn covn u~~ r~~ M cn~ c~~ v ~ r~r~v r~cnv mv i
Q iUIQQm Q QIQm mm ~mUm Q QD m D DU U mU Qlu!Q Q QQ Q mm Q QIQr~
~m
_7g_
CA 02290738 1999-11-19
WO 98/53103 PCT/US98/10561
TABLE I (CONT)
M
iI M r M N r M O M m O ~ Cfl~ h~ M r
C ' 1 O N h V O n
O I
OO ~'~C O O 00 OO r D) ~ (D ~ 'r V Dr O dd O ~' O ~'O O ~
I 1 OO f O N O~ O '~f f ~f O ~'( M r ~
C O ~M f M O CD m
( ~ C
O r 0~ O NN N N ~~ d Or r nr ~ ~r r Mr r NN N -N r Q7N r VN N
4 1 I C I C O O O ( O "O
rC
1 ~00 T rr r r Mr N rN 071~~;'fD(Or T ~.~ O ~ ~, dI~ OI n M
~ N N ~ M N O 1~ N 0~!O OD M r
0 ' (
O 0 O0 ~ Dn D n dN ~ 01 O 0N f f~ O f'r t OO O rN M ~~ ~tNN D r
C C 0 t IC t M O d 1M 0 O- ~ 0M ~ ~ C'(C r t " C
O M O ~1 a C O OD
!G
f ~ O0 ~ OO O Q7'~M D Or r f7i'N Mr ~ pr r NN d NN r CN r VD r n
M dV'h ~0 N C ( C t t ( ( I
I n 4 0
0
I
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N UC ~ N C ~ ~ N ~ O ~ E
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O v Y Y~ tbY Ut d 0N LLNr M ~Y ~ O-Y :~~~G ~ OO O NZ (nEt p U
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M C C CLLn ItI-Q N
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!9h r~NODNO~IG0C00)O tl7rM 0 ~N ~ IW V OO COa~O ~ o~O~~ ~cDN t~~nCON
'~I,,-I~.Mn r-0~- t~f~.QOO tnr O InODO r-O O M
M DDM COO~ M ~ COO W f700O (D d N r-M r r-
r m-t17 O c00~OD00I Ol!~ ~tLnM ~
1~ ODM tn
O t0CDd r ~h 0007(DO O OtnCO _ rapN tn
cD ~Lnlf~OM aDO~ tnLt7plyCD1~O O C'(DO
d ~ (DO O OfD!~d NO r r-C'r-rr r NN N NM V VV'Lnlf~GOCDrQ)O rM I
InInll)CO~CO~ d0~N r MM 'cf ~ ~ ~ XX >-DD V I~
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CJX XX X IXX X X XN J 'JJ J ~~ ~ _~ > >~ ~ >> ~ ~ I
41
I
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U t._~ N E E ~ C Of ~ 11 ~ N Q 1. ~ C EC ( 0. U tO ~ G
O t ~ .CU Q '~ ' tcDV '~ - tt ~ .-.D7 7I~c0r
'' ~ d ~ ~~ ~ ~v c0~fu I
~C ~ n
v . ~~ <Dt0M 1~M f'( M t0tD UU U UU U U0 0 IUU U UU IUmU U lma lmO
tD I U
Q m QiQm mU tll Qm U
U mm U
-29-
CA 02290738 1999-11-19
WO 98/53103 PCT/US98/10561
TABLE l (CONT)
I.
I
p t nni M NM M( OI V ~ ~~ r O O IOIO OI
I 1N NIOI1 0M ~N V'O 0 0M O O M O ~'~'i
O f
O rn N O MN O ~~ ~ nh D D ~fN M D~ D D0 I ~r M d'M n DV O DM ~
O IIM O I I t C f M rM ( 'C O 0~ ~ MOr" ~I ( ~C c f
C D r OM N MI rr' N r
N r O ~ NNIrCOO M rr u7~ ~r M's~ O Dl7N ~00N ~O ~ ~~ ~ ~~ 0
~ r ~ ~ 07~r-D ~~ I . c ~ I f 1 a d
r a
H M COO r ~ Q)N N Mr D l7N n M rO C 0n M O~ OM ~ rD ~ MD I~rr '
O O (D ( t NI ~ D r0 0 IO M ~~700l7O~O (O N (l ~ ~N ~
N 0 ~ ( d O 0 C f f d' ~O I
0 1 0 O
Q.~ ~r n ~tOO ~ Ov V rr r r rO rI~IM N NN d'MdOr~f7r r rr r rr(O
r I n r NrCp ppl(
rI
N d
J ~ Q O
)
z~ N- _ ~ a
o r
~
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~
LLI M CJ~CL .a .ct3
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ifl L p
lJ
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~ J ~t Y cts
LLIO ~ CLtOn~ N O ~ ~ U Mp
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-
O O OC v07tOn~c'aN - :~ L ~Q ~.
V
(Ov _ Y vc0caa1 . N. ~-c ~ c0 ~cDa~ ~ r O
'Y' f v - ~
~
~ a. = ca n.c,...c c oo Y E'o c_. Ua~m ~ ~ s_
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~C~ y
O U! c ' ~ Yc0 Q~ ~ O f~ c0~ NN C "-F
~
' N' .O Oz O tf~~ O N fd~ V ~ C ftSV r UC ~C C ~ C
Q C C ~ E = ~
H--d OO ~~ cArv. _ ~' ~ C 7. 47 NY 'a fOO U
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C 4YY Y IN -~ n-YQ '~Y rJ c cr o ~X U ~ ' Q
~ CO ~ '~ ~ ~ ~ ~ Q a c '~
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a
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(OION I~'r ~ (OIO40~'~ t~00hN 00(DO ~ ~tn'~tMd'M rL~17~ ~0000 INC CD
' M
C ~ (fl N N Nr C II 1~(DM 000000O O fDr OD~fDO (D00f~CDCDN 00r C C'(~CO
VN N N N~ Lf~rN Lf~lf7r r r OO M r1~GOON N MM lf7f~O)O rM M f~M O
r h InInO ON ~t~.d0r I I'CO MM ~ d'~ ~ >n~
ap(p N C'
~ pO O p O N (DCOO rr r r rN INNN N MM M MM M
rN INN I~ I
C~D JJ IJJ IJ
C
III I
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I
T .- v ~ ~E v ~r E c m .n-o ~c Eo ~
cu ,~ r - t'_ t~ _ ' yC O Un Y ... 4 7- ... E
~ u7CO CDf~I~1n ~ n ~ - .Wf7
I cD t' ~ 1~cfl~ 1~ r ~ t' I~f~ 1~
' In h
L. 7I~t'1~~f ~f7lim QIUm m mU Q QQ O DtLU mU Q Um m QD m QU D
u C I m ~ Im
aluJ QIQ!QQ uJ
-30-
CA 02290738 1999-11-19
WO 98/53103 PCT/US98/10561
TABLE 1 (CONT)
r M In d0 d' O f~ N r ~
C N 1~ M V 1~. 00 N 00 ~ O f~ d0
p tn V' C' CO O f~ ~f N CO O CO N N I~ r
O N M tf) N QD M N N tn eI7 CO p N (D M O N
N 1~ ~f' Is V' ~ fs O OD M CO f~ V' ~ C ~ ~ N
r~'rOpCOOp(DM MrlOpp(pM N N
O tn r '- r r [~ d' O M M N I r N r (D O p
a N M In N (O N N N ~ lf5 M r N r (O (O r-
Z
w a m
m U Q
Z Q m
I
O ~ ~ Z
Q Q
v
f O U
Z
n ~ O
m
Z Q O
c°"n ~ a
n. a=. m Z
a~ ui V ~ ~ ~ a
a a c~nv °~,' w na. ~ o~ z
° ~ a cn
cn ~ = O V Z N Q
o. s a~ .S v°,' ~ O m w
o a~ c i~ .c o ~ I- Q E- U
°- ooQC=~ ~~ QZ
a~ Z d
d U ~ .~ 'c - ~ ~ ~~ ~ U a c~ Z = J H
~ .c a~ o o . ~ o o .S >- e( m ~= C~3 Q Z- p Z
- cu o o N ,~ x i en ' ' ~ ~ -
ea=,cQ o Qm mX~ C ~ ~mYC'3Z~ Q=Ot-aX
d c0"o ° eon E ° ~°' ~°' N ~ oW . .E c'nv U L~ j U
Cj Q D m ~ cO O
' m
d E ° m ° '~ z z z ~ ~ ~ o p '°' j ~° ~ = f- ''c O
O O ~
c~ ~ co~~ ~'nnn o>a .n nUal~~-~-=~mNo~ma=
o~
r
O
N
C
O N O N ~1' r O N ~ O O (p ~ ~ 1~ OD O r N r O O O
m O (O M_ (D (D V ~f O ~ O ~ ~ p~ O c~D ~ O c'~~ ~ ~ O ~ tM
d Op N r M ~ P~ M
C O r M CO r M ~f O ~' 00 ~' ~ r f~~ r O r O CD ~ p O O
41 CO f0 CO cD h CD OD Q~ I- O tn N OD r p p r O lf~ r N OD p
(' > > > ~ ~ X X XI= = X J ~ ~ X Y ~ X X ~ ~ ~ >
d
r
ea
c I
'O
O
O
U
;°_ c c ~e co .c _ E c c ~ E c o~ E c N M v o~ o r
~ h ~ f~ In In h h 1~~ h f~.' 1~ (~ 1~ f~ r r r '- N N In CD I f~
au.IQUQUUUUmu..ItiImaQC'3C'J C7C9C~C3C3C'3C'3
-3 I -
CA 02290738 1999-11-19
WO 98/53103 YCT/US98/10561
Muuse .~rrUy
In the mouse array according to the subject invention, all of the unique
polynucleotide probe compositions will correspond to a mouse gene of interest.
Mouse genes
that are represented on the array are key genes, by which is meant that they
have been
reported to play primary roles in a variety of different biological processes.
Typically the
mouse genes represented on the array are genes that are under tight
transcriptional control.
Genes of interest that may be represented on the array include: oncogenes,
cell cycle genes,
apoptosis genes, growth factor genes, cytokine genes, interleukin genes,
receptor genes, and
genes associated with different stages of embryonic development.
In certain embodiments, of particular interest is an array having the
following types
of genes represented on its surface: oncogenes & tumor suppressors; cell cycle
regulators;
stress response proteins; ion channel & transport proteins; intracellular
signal transduction
modulators & effectors; apoptosis-related proteins; DNA synthesis, repair &
recombination
proteins; transcription factors & general DNA binding proteins; growth factor
& chemokine
receptors; interleukin & interferon receptors, hormone receptors;
neurotransmitter receptors;
cell-surface antigens & cell adhesion proteins; interleukins & interferons;
cytoskeleton &
motility proteins; and protein turnover. In a specific mouse array of
interest, the spots are as
listed in Table 2.
The mouse array of the subject invention finds use in a variety of different
applications, where such applications include: profiling differential gene
expression in
transgenic knockout mice or other experimental mouse models; investigating
processes such
as embryo genesis and tumorigenesis; discovering potential therapeutic and
diagnostic drug
targets; and the like.
-32-
CA 02290738 1999-11-19
WO 98/53103 PCT/US98/10561
TABLE 2
I
O ~ ~
C Of N ~ ~ ~~ c ~ f~O N G ~ O
D 0
O Qf N ll7r MtDM ~DM OtnO r .~f~OM ll7lf)tf7(D~ ~ CO M M l~tl~ M
~
r O r O rr 1~00sT00NN N M ta7M f0IniwO rO M fN COr r r (pLnr
r r ~ r ~r CprODInIn~ O'O 1~h O ~~'r ~ll7ef r CD~ r ' ~N r
i
~ V'f'efr CO~(DO rCO~ C~fCDN COCO117n t17 ~In MInM rM
H ~
4 U7 CDN f~O~'Inc0M O InN i'~r _ ll)00O O MN M OCO MO cQ~ ~M tt
CO (Or (OrCON CInN NN r CD~V COCOr (DrN r I~O (Dr 00r CD(DCO
m
V
C_
V
U
E s E s cv>.o v ._~ o~~ _ c E m a E 'o ~ c r a~.om
~
WO M tl7N Mr N MN N rN (DM rC ~ ~~ ~ tnM N ('~r M[vN r NM r
'7
Q U u.luJm u.tLti.UU t,uu!u.m D tLU u.tiu.u.mU U ~uJ ~uJuJLuti.~ ul
c
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pp L >. O O CO .:_
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U
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' ~ 07' ~~ C llU E '
O C O ~ ~,.-. N cC C N d
~
p Nd V ~r N j'~ LU s .C C
U . Uu'd ,C > r > N L O
C ~ ~ 1.'L. c0(O .... E~a O ~ n..
~ m p .
O ~' NC~C :C'~ p~ Nc0U '~r ~
L lyLLp Of r C .p U~J.C tC41f U U dLL
p ' C O ua ~ tt1
f
a _ ~ U a .cr a~.~oa c c~.c c_~ ~ c .~ ' aU
I
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N U
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U UC U ~~ CU L ~ O N U pI
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O CU ' ." ~ ~ p7~ O~ OC 0- O UE U
- I
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p C j ~ C~0~ O
Z O ~ LLO~ U ~ y a Z ~ ~ NlC. ~ C 0 , c .-
p 9
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U~C .~~~id . f--O c0d LO J U N L
- ~
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L1!~U1~ L C_ C_.C'~ ~ fy'O y ' ~ ~O d N 3
O ' O
U U ~ ~~ ~ O Ol1.a .C:cC CN O ~.q) ;~L U Q7p~ N
~
O O C ~ O p _ O 'C~ _ OC
~ O U ' a a
O - LC O ' ' N ~r In~C .~(C
~' L ~o a ~ a a o r r V C r C O d
U 47 d ~ ~ ~ ~ ~ ~ r C N~ ~ O
O
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~ ~ ~
l~t17Y C . ~ C lU. cflO~ N _ JU y V~ Y -~~ -~ _O E- O p OO
Z ~ 7 _ - ~ 7 ~> > O'- C
~ O ' r '~. '' L ~ C O~ N ON O M UV V d U
~ ' C O ft3Nd O C~ U ~ ~ ' Y~ _a Y ~ d
d ~ NU
N
C E ~ ~ H UQ d E0 ' '~ Y Q DN p Nd d ~Q D mU 7_N_No a~a~v
d ~ cu~d ~ ~ ~ m ~ ~
C7~ c U c~OU Z u.U ii,C~C~n.C9Uu.~ >c c JO U ~U ~~ ~ U =IYiQ
~
Y
C ODOD N I~NO r O N MGOCD LO07 V'Q7lI7 M
f~ f~
M 0000CM ~ O~ ~ OO ~ rtTIwON M NN N d0COCDO O
07NM O tnMfDCDi ' I C 1~OD I O CO
N ~ I
M
C' (ON h O CCQ y ( r(O MN 1 ~ n.
O N O Ir MM (Dl(7I ~ tf M CD
C M f Inr ~' 00pN ef M ~ l(7M J ~t(D tpN QIN N
~ l!71~ d' ( CO00O ' I ( '
~ N
~
_ _ N l17f0!D!~00 ~ r r00N ~LO OtOIn1~ N
~ ~ J I I ~ ~ ~O >~ > >> XX X i ('
I I I~ 7
XIXI NIN
C~d D D O D ~ ~ ~ ~~ ~ ~.~ > > .,
-33-
CA 02290738 1999-11-19
WO 98/53103 PCT/US98/10561
TABLE Z (CONT)
I
o~ S o d a~ r~m d ao o
~
C O M M Nr O I~Nd C' M r O dD
O f0O (D d1~ d ~ M O~'COdN M 1~O d COdDtI)O Inr (DIn (D
In MC -'h N;
d r O N N tnrr O rM N rM r d r [vd ~ O Q
In InQ) I~ r (D CD f~r (DCD'~r 00 tn
V' (D ~ M (O N ' l0r ~ O~ OI
I~
d
O r r M NO I~ ~ f0O ~ rd lC)tl7d V' N O GO'~COO (Dt17 tn
O M 0 N~ ~D i
M
i
O
0 t\ f~h r h QIIn000 I O d00r r O CO1~OD00N
. N M r rIn N N M rh N rN N rm r d ~ MM r (prtn N
I
00
d1
C
_
O I
O
U
a
;. ~ a~o~ . cn a~a~s .xs c vE a~ c m E v
_
w ~ N (O rN _ d N l0CD(O1~lf7 CD1~I~CD M h U7t~f~IntnIn Vd
Q m Q U Um Q Q O muJO uJU COOuJU m U Om Q O mU QU
I
N I.C
d m
m C
O
X N C a0 ~ ta0 d
O ~ a X Ud
C
7, ~ 7 O C1 UO '~
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cD y ~ ~ Q 'C ~d O
E c o a
n ' ~ _ Y E vY
.E
, a m
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t U 7~ N O
L O C
c 3 ~ a~
~u
_ oD ~ ~ a c ' cW a
m
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- E
E c o
c c
E y ~ ~ c
.~ cm
c ~ r
' ' o ~ a aio '- y
o '
~ ~c ap o v~ ~ ~ :D~y
~ a
~ ~ mU a o cu ' c ~ = a c
~ .
' ~c d - O ~ a . 0
N ~
d ~ ~ o c a o~
_
a, Z r--' c s -- Q ~ '~Q aoo ~.I
a ~ ~ 'c
~ c a ~= a~ r p E ~'~ ~ a~~I ~It
'a~ Y o
c a~~ X aY ;
. a
v
00 ~ Q U ~ ~~ ' ~ Q -om ~
~ ~ ~~ o
O E o '' o La ~ N ' ~ ~ a ai i
d . =
. ~~ E o .D_~ o ~ ~ _c~.~ ~
- o a N> cn o a ~ ~ ma ~ Q ~ '~ a
~ g ' ~ i o
N ~ m
- N ~ ~, . o o ~. ~
~. ~ . ' N C :r
L C
C
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U a 'a ~
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?
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O~ r O
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~ V ~ E C O C I
j I C
N
d of N N_ ~ ~ C ._'CO ~a N Q O ~ O sQ O N, ~i O
~ ~ ~ ~ m d ~
~ N
, ~ ~ mH- ~ a N ~ a , C3cp~ U m~~ M i o >._ ~t
'vio X ~ ~ ~ U a %
c c~
C ~ , o Y ~
r v
c
n
C C~'- U j~N N Ud j ~d M CN U wIU VM p X E~ .!N
E tn ~ j O a O! E
.
~ p ~ m~ u. E v~Yo , a~~ ~~a~ ~ cr~~. Q ~~- a~
~ o ~, c ~, d~ X I I-~ E
:
C~H-Ns u! mm Z n.ulaU F-ZQ C~1-> tL.--UIXIZr a.a.UQ CL:Z
v ca, c m coi cv
I
I
I
I
j
d O CO N(O ~7 ~ 1~r~ N _O O Of~(p ~ f~COr Inf~tnM
d ~ N d MO CO00CO ~ GON M COO~
N
M
O)
N
M d O ~p O C)r 00r O rI~ COMd OD (pC~0NM r d O~
O M M r r
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I
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C d CD d~ r M O In1~r CO M Md (pN t'~1~CDh a0t0
NIn (~ ~ I
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d O _ r NN N ~-N NN M MM fDODCOn [vO tnt1~tntnInCO
1 I I(D
CD
C?01D O JJ J ~ ~ ~~ ~ ~~ ~ ~~ fn CO~> >O X X XX.X
X
- >4-
CA 02290738 1999-11-19
WO 98/53103 PCT/US98/1U561
TAE3LE ~ (CONT)
__
N cc ~' u~
c r ~ a~ ~ a°~o ~ ~ a~ aM~o ~ ~ v v ~ ~ ~ ~ w o> m ~ ~ o~ M a~ N
°o ~ ~ I r
O r O M r r ~ O r M r N r O CO N r O 1'~ N O~N r M
ly .Q1 cp N r 1~. is C4 M ~ 1~ In r ~ C~7 N ~ tn (O r ~ ~ ,~ CD r r O7 ~.;~ r
H tn M 00 r O In '~ O N r tf7 O M f~~ In f0 N M CO N tf O O ~ OD 1~. ~ O 1~
O ~ ~p p7 lr7 tn r O !n r M O N N C' r N (O O CO M M fD
d. M t!7 M r OD tt7 ~f r M r N r f~ (O N r N 00 N I OD M r r M C7 CO h r W
N
,r
l0
_C
v
O
O
U
.O U Y f0 f0 C U .Y ~0 d
(a0 tn r tD tp M r N In tD ~ In N V f~ N ~f il7 cD M 1~ I~ N N h N ~ 1~ M
Q m U U O u. U O D m uJ m Q O D D Q m D O m D O A m Q Lll O u! D
E °'
E ~,>°'.
cu ~ a~ -~ ~ c
E ~ m c ai E Q,
m ~ ° ? ..
o i~ a~ -- m o
Q, 't 'ln r lA
n. a v °' ~ o
c
. ~n ~ a
o a~
a
in _o _ m ~ iD ~co ~ c ron
Y (6- tl7 ~ N I,aLJ Cp U >. .~ O
a~ ut ~ c o - ~ a ~ E
0
U c a~ ~ .aU y ~ x
Q o o ~ ~ J ~ >. a~
w > ~ ?~ .n a~ a
"~-. a y o v~ o c ~ ~ m ~, ~°n c _ wo
"c X~ ov rLa ~ ~ c
as o N c ° ~c u. as ~ N ~ ~ ~ a v
u1 a a ~ ;~ o c N ~ .~ E o~ °'
<o 'n ~ U o ~ a ~ ~n y ,~ 'o .-. ~'.~' .. a >,
Q N ~ ~ C j Q C ~, ~ Q. r r o ~ C
m ~ ,= ~~ o mi o E r M
c0 V O O ~ N 'V O ~ .U O- N O E ~0- U Q O ~ ~ ~, ;C r~ tL
aE,S-oaca~ a~ ~_.~--ovxc
N .tn C N CU1 tin .C .~ ('~ _O c0 C C t0 ~ .~4 '_ m v N O C = .~ O
Y U ~ ~ ~ >. N C ,- C ~ ~ 07 E ~~ ~ E O m Q Q C .Y L ~ r O' N
_O ~G d ~ r O E U U LL O
N t ~ O ~ C O .O V r V1 O V f0 C z (/) C ~ .C U I~ N C
V7 V C_ ~ V N O a .~ O7 cC C ~ ~ C V O
o. U E a~ a m ~y . .4 ~ . s .Y c
c m N o .~ E °n '~ °' N '. m _c o o ~~ ~ ~' ~ o a~ v ~ a o .o ,t
~.
~e d a L Q > ~ ~ a~ ~ ° c m w ~°: .- a o ~°, a~ v o E ~ r
o °- a d ~ a ~ a ° v ~ a~ u~ a~ ~, ~ .:°_ a ~ _-- .5
Q '~° Z = °' ~ E o ~_ ° ~ N N E °. S c - ~ v
c c a~ '~ ~ ~ ~' '° a ~ c
a~ o U a , o N a, c 5 m a~ ~°' x X v~ ~o :o ~ ~ .p o c ~ ~ a U_ ~ x ~
c'a
E~,~ CU a .~_C~pO~.E C C~_-I' :OUP U , O
_>. L ~ ct1 O O_~ V m - i._ N Y iC
Z Y O N iC ~ d7 'C Of S' E O' V ~N O fZ 07 ~y c0 .a m N O .a~ a C M ~ N d ~ O
C M
L r O C ' O ~ Ui U d M Q
c a Q a ~° o;'v~ N N a v Y ~ ~ ~ Q E ~~ ~ .~ ~ x z ~ ~' ~ Q Z ~a ~ ~ o
d Q Z m ~ cnl c o o ~ a ~ o >.m o ~ Qlu. ~ ~ Z U Wn c o a~IQ
C~ ~ D U v ~ Q U E a uJ -~ D tO J n.u Z N ~ t- Z n. _ F-'~ f- D U ~ a o ~n Z Z
, CJ
Y i M M m M M
N ~) N rl0 ~ r r OD O O ~ r GMO O ~ NIN ~~ O 1~ O N N
m ~ M ,p, fD I'' O r N M Iff Q CD O M N ~ f~ In I~ ~ O tn I ~ N
C ~ 1~ ( CO O ~ CO N r ('~~ C~~7 ~ ~ N O N ll7 1~- (O t~ ~ ~ ~' 0 O N N M ~ I
~ I In
_ _ N N M tff tn O) O~ I
X r~~ D DI~ OJ J J J J J ~ ~ ~ ~ ~ ~I~ ~L) (O~(O ~ _ ~ ~ ~ X'X~X
-35-
CA 02290738 1999-11-19
WO 98/53103 PCT/US98/10561
TABLE 2 (CONT)
M t~ff 117N ~ O O I~
C M M ~ M O7MO ~tM d0r IwOI~N M COtt r 1~C1O r M 1~O
~ N r ~N Inr rf~M rM r r Inf~O OO N - m O r I~rO M CD tl'7CO
f~ GD
~' I
N
47 CD~ tl7O (O~,:~~GOCDrCDr N 1~Lf~r nf~~ N 07V r M f~r lflC~ 01~fl
07 ~
O f~N In~ ~ f~InO M MN OD~'N(D~ ~O tf)00 (DO O tn'a'r N r COOp
N N
~
O f,~ CDN ~r r (OON N d'M h O tn1~00GOO ODO O CD r r f~O 1~O InCO
r I~
In N ~-NIntnr rIn(pInr COr ~N f~~!'V'r r CDr Car et[vr r CpN
CO I
r
I
d
.
~
C_ i
i
'
O
O
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~ U .~Y E EC ~ C~ Y Y CC1p~td .,..N ._,'O E YO c0 L C
E
v.I~fsM C'r1~~ COll7tnN rN ~ M C(DCDf0~ f0h h (G N ~'N<OIn'~ (Df~
N
Q O ulQ m mU U O UU U UU Q Q QD Q QuJQ U Q U t~U u.Q Q m m uJ
Q
'
o -o
V .-. b N
c~ N c_ cd
. tI7
7 O _ z _ N
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n
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CA 02290738 1999-11-19
WO 98/53103 PCT/US98/105(1
TABLE 2 (CONT)
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CA 02290738 1999-11-19
WO 98/53103 PCT/US98/10561
TABLE 2 (coNT)
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-38-
CA 02290738 1999-11-19
WO 98/53103 YCT/US98/10561
TABLE 2 (CONT)
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m00efI ON t~N '~tl7Ot0r ON Q7_ ~COc0r~ N (OI GOOM (O~O CDO ~p
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O C In tl7 ON O ~ MQ r r I r N N N N~fM ll~Il700GOCD40O ICD
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-39-
CA 02290738 1999-11-19
WO 98/53103 PCT/US98/10561
TABLE 2 (CONT)
I I
O O O 001~f~ tf)00r M Mr 1~N f0 COt01 ~ O MtnQ
r M 1~t~ (OQfO r d'tn V ODfD 0' O
In
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~ I d0' ~O r
Q r r OO N r0 O r rN r ON r O r1~O NjN r ~ V r~ CO~'r r
r r ~ Q ~ ~ ~
H r ll7In~r (ON~ ~ N C1DM ll')~n O tDthM ~ Q7O InInc0(D a0O tnM
' a0Mc0t~ N Q7 tD ~ (DN c0
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OD O InM I~~ M CD M
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I
41
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v M M re rt0M tl'tn r Mcf~ ~ r N V NM f0~ r rN
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C N O N N OO I~(~N r N InQ 00OM f~(D~O~ M N N C'O
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C31~I~ fD> ~~~ X XfvIXX XX iXXX X X XX IX X XIXIX O D DD O OlflO
I> IX
-4O-
CA 02290738 1999-11-19
WO 98/53103 1'CT/US98/10561
TABLE 2 (CONT)
I Of N O M .- .- ~ et N ~ In
N N M O O r GO tl~'7 CD r N ~ O 00 O O 01 O f~
C ~ N (p r.. ~p In t0 Iln r O N ~ ~ ~ M ~ r (p tp In ef M In N ef M N M N U7 M
ap r M N p O ~'- f'~ ~ O) ~t O !1' O N N In O r r M r r I~ N r O r r O N ~ r
~ M 1~ GO O O C~ 1~ I~ r p r r r CD O CO O' ~' r O V' ~ In ~ ~ I' N ~ r p p
H N O r In ~ h~ N OD (7D N Q N ll7 In I~ ~- r N O O CD O V' ~ tn lI7 '~ N In N
f~ 1~ I~
O tn ~ In tn r N N r (p In ~- N CD N r tl7 00 V' M ~ N (O O r O N O I~~ O O O
N r O
a ~ r M N 01 CO r f~ M ~' In CO In G~ O O ~ O N r r O ~- r ~ N r N r r ('~ Q7
Wit' r
41
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-4 I -
CA 02290738 1999-11-19
WO 98/53103 YCT/US98/10561
TABLE 2 (CONT)
C7~ (OCO ~~ ~ et'Q'(Ol_n~ r
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WO 98/53103 PCT/US98/10561
TABLE ~ (CON'r)
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CA 02290738 1999-11-19
WO 98/53103 I'CT/US98/10561
TABLE 2 (CONT)
C10 O O ~' ~ OJ ~ O '~ r' Cf r In N ~ N r N
C f~ tt7 O N CD M CO M CD OD r t(7 O CO 1~ C10 ~ N
O In f~ ~ ~ ll7 M r CO ~f O ~f M OD N f0 r tPl f~ N N I~ 1~ fD ~ ~ N r O O N
r r N ef r N O r l17 N M N N M O ~O r r N N r O M O 00 N CD V' '
tn O O ~ tl7 C~7 r (p f' ~ ~ '~ r r (D ~ r C'7 f~ (~ ~ ~ '- ~ N ~ ~ In 47
O N In 00 M 7~ O N (O tn r O (D C'~ N 1~ M M M M OD M O CO h OD N f~
O N ~f r N N O ~ ~t r (fl r '~ CO I~~ N N N ~f O O '~ O 1~ N r O N f~ CD
d r r N M r r 1~ r In r tn I 1fl O Q7 CO V I r r r N r 1n tn fv CO r lf7 M
d7
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f~ In 1~ 1~ t(7 ~= f~ N r C f~ M tn r r M 1'~ CO (O N f0 ~ h In CD ~ CV CD O
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n~~'~na~°~ ~'~~oi m~'° ~v°~cNOO°m.~.'n. °E
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N O = T ~ _ ~ a U _. -: C " a O > U LL .U m U N L a I N I U Q = cn C~
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47 J ~~ c9 T~ ~~ EI~ E'~mCCM ~~QQ CI-J' VIQ >.C
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m 1W OI N CD r f~ r (O (O I 01 N In 1~ M (O (O r r N r ~ tI7 r O I~ CO r ~ O O
Cef Or~O~l1 I~GOO M'.V'(O1~ CO (DMIrNM ('7MMf~~MaDi00IriM
CA 02290738 1999-11-19
WO 98/53103 PCT/US98/10561
TABLE 2 (CONT)
O 01 O~DCp tDI~ 0pNN COO cD
~
C CD COOCD0 f~Nh ~tCO Inf0N N 1~00l('?d'~ r r N Md0r M r~ trO
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r '
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s '~M D ~ Q
00 M ~~ N a0stO~ Nh ~ ~ O~07~ O~ O IODC O cDc~t ~ 07~tM O
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a
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:O O
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CA 02290738 1999-11-19
WO 98/53103 I'CT/US98/10561
~CAt3LE ~ (COt~IT)
r In ~ r (n
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C M M C~~ I~~ M f~ In CD C7 O r ~ ~ O N V' h V' M M O CD 00 M M In ~ Q) In dp
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U a E 'O C of a U G. C ~a O .:b .:a N O ~SI CO ~ N O p ~ 0 N
o ~' s ~ ~ ~ r c Y .a ~E m U ~'-° t .W'--' a t t I o E a _o c a
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C . t U _~ U ... fp
C
. ~= ~ d p. N ~ ~ O O j ~ O O p d ~ 'a a j. ~ E ti7 ~ VJ E
41 (' ~ (L~ 7 E ~ ~ U ~ r u- ~ m ~ t ~ ~ Q) C E O) O~ p r ~ ~ .U ~ ~ U d 47
E 'cc E n. ~n D c I- rte, o a a~ ° ._ m ~ ° o o ~ c ~n o~ c
Z -p n m o °' o .° Y o N ~ o O O ~ X C ~ Y Y N C Y ~c . a ~ N ~
~ . .n. Q ~ L
y C~ co c .- a a c ~ a ca ~ ~ -- -o .E ~j ~ ~ ~c .c a '~ .c 'c I ~ ~ °~
~n r, a~ N o N
C ~ x ~U I O ? ~ r ~ ~ O ~ ~ .~ r ~, ~p Q ~ 3 ~ m c0 ~ ~~ ~ ~~ E ,~ ll O C U
.D
d c0 Q 7. t 'O 0 t N U E ~ V N. = O a N O U N LL ~ of u1 E N Y N tX tn N cU ~
7 X
C7 > a U U .S , a. S > ~' o_ ~ Q D ~ C7 x S C7 S 5 C'3 a c ~ ! Q g m uJ ~ a t-
~ CL (~ N
I
C r M N ~ M ~f Q1 N CD f~ r O O tn r h M N O) O M cf ~ I~ CD i~ ~ O V M V C'~
t1~ M
m CD CO M I OD r r et ~ N r r M ~ tn N r Op (Wp CO CD ~ N M In N N d' In tv O
In (p
C M O O . O O CO CO N M I~ M N 1~ M V' r lf7 l(7 In t!7 In ~ M Ln N M 00 r ~
p7 ~ ~ O O
M CO 1 I~ f~ D1 O O r N N N N Q In f~ r I N M f~ fD O r r ~ 00! Q)
C~ X X X~ X X X X X X X X X X X X X X X X X XIX X X X X X XIN X XIX
-46-
CA 02290738 1999-11-19
WO 98153103 PCT/US98/10561
TABLE 2 (CONT)
Mr ~ N 00r'InCDO In N (D !1' N
'.M00 ~ N f~ ~ MO N O NM h h r O r- r IvODI~
O N CD
O Mr MCOGO~'OM 1~f~ (OInI~6nlf7(D47 hO tn COIn~~ M h ON O rM
MM f~r ~-I~etr ~-r r ON - r N~- Nf0~ r ~ (pr r r
r~ ~ ~ ~ ~ ~ ~~ r r ~_ T
(Dt~ M Iv r (pnr 1~,-ON (D N W
Q1~M CDM O O
N QW MO cDO CD~ O cD 1O~M c0- cDO 1~C~tnMO r-Nr aDCOe-f~InMO
O D Nn MO r M
C.(OCO NCOV M OfdO f0In N f0Q N N Nr Nr N rr r CDr Iw!wt0r 1'r1t~
MN f~r r lClInr ~ r r hN r r
m
r
A
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O
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'flU E C t ~ Y ~C t _ U C _t 'OO QfN C N E t
N h ~ N MN ' ~
v.~0'~ f0(O~l'~Y~fcf~ tn fDrf0M C~~'N r~ 1~~t ( ~..In V r
a sa om U w ww w a U ~a o Q mm wu.m wU ~ ~w ~ o ow m ou.
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t cC ~ .O-N f~.~ O > O t U v C G.
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0 d ~C
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m 1(D (D~ O N rN O ~ 00tf1i~OD00InO ~00O OO Inf~M O N Mtn~ ~I~
~00 ~M <DN r1nr tD O (OM r NO IntnN twf~0700N I
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C3?->- 7-X INN NN N N N NN N N N
-47-
CA 02290738 1999-11-19
WO 98/53103 I'CT/US98/10561
TABt.E 2 (CONT)
'~~COD ~ UOD O 000 ON (pI~ r_C7
O rm O r r
Ctn Cflm (DCN r fv M r Oh M NN N f~et m 07N f~COMO N U7M 1~mN
O InS' m M 1~1~ m N(Om O rr r rr O rN
O rN r rr ~ CO InN Or I~NO'r 1~I~ (DI~In~'r rlr7r h!~~ CDr
~ 40 (O~ ~~ O O'
Nm I~~ m CD(O1~~ InV'CO I~Om ~tN N m ~N O N rI~V'MCDr f~tn
N O rN
O~p ~c,p M etO c0m N m Otl7M ~fO O ~f7m O YM CO'~OCDO N~'~7~O~
I
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dU pO Y Oof~ () ~ Qrtf(nLLZ c0~ m ?.Ctbt O ~.-D O c0~ 7 ~
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cn ) l
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c~; ;xx >-ao loo - ,
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-48-
CA 02290738 1999-11-19
WO 98/53103 PCT/US98/10561
TAE3LE 2 (CONT)
i
~ ~V7t17 O M1~tf7 a0 ~v 1~ r N M~
' O M Nr GD rLn
C CD O Of~'~ OO C ~ Mr ODCM lI7rtnC7ODtDf~. 1~O M r O (D(DM COCON
O M I ~'(DM MN N f~r OV N O~ N MO - N rr 1wNOD
N O rr
_ N M rN ~ ~~ ~ ~ ~~ ~ ~ ~~ r ~r r 00~C!3
.~ 1 ~ N ~ 0 M N N 0
wlM ~p(~N.,r N ~ffN V sfN O aOft0OO C0N N- t~ f~~ f~O 0~tO ~N
O O M rM r OD00O ~fCO N 0O N ~M V M CON ~ M1'r ~Dn O r 0O ~ COOD
CD N
O.N N rN r InM r Inf~N (Drr N Mr N N ~t(DM 00M N (D~tQ707rr U7r
N
r
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f0 C ~ ~ E~ C .O ~G ~C E U Y 'D ~t~ U~ t t~ ~ c0E C c0t
v D C Cpr-~ f0tl5I~(DInMh I~rN r fDM InlClN-Q1~NN
M r (O( M (Of0M CO~r w w wL w n mlin tim U ww w w ow a UU
Q m tLWw n u.tiw w ww w w ~
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s ~ c~'o ~ y c ~E .E ~ 00
O N r
p O ~ O C~' . N
w E E ~ ~ ~ ~U O U O L _
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c c~'~ .~ M Q T U a ~ ~~ v~o
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O ~ O ' ~ C
Z 'O I ~IS1 ~XN = v O. f0C~ OfNc0~tC rO ~ EI m ~0 E f9_t C ;O
p~I p T O OD .
N I , , COQO 1nLC E O~ ~ O~~C ~ CN N ~ ~~ ~47
N O ~ E
G Y I 1N X L O N 'p ~ a T a~ - ~~ . CLOO U nO d C 'C= .O
cC c0M ~ ' O cb
nn ~ S.m ' o ~, p cO F t~O
CSZ:YIC~IUU ;C7UU f,~U w~ U OU D C~~ U ~ ~cnu7m:Ym UV)n U Wn If-(.9Q
I
N
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yk ~ Y
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l0 rO ~ IONN ~tM rr CO0D~I11NM~ Q)N OINCON~'-O ON ~ ppM~ 00~M
fDlf7 0COr NGOr C)OM 01O~ O rM O - M~ N
N
d0 N N I~ON N ~ tn0 O t0~W QpCDLn~ M
m O M O71~MN M ln~ON 00(OO InOM h.r'I~ODN CD~OCOC'r p~r aDr 00~M
OO N~tM ~!GOr M ~'h M (ON tn(DNC1O In MO
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O O rr M M M~.O rr ~ ~O O ~ OCOP N r N tI7_ ON
IJ > > ~X X X XX X ~I~W ~~ > > >N ln~J
CSI nIQ J ~~ ~ ~ ~(n> >
N
_c~9_
CA 02290738 1999-11-19
WO 98/53103 PCT/US98/1U561
TABLE Z (CONT)
r ~' ~ O CD (D O o M
O ~ ~ ~ i~
L.Vt~ r (O M O ~~!7!wCD GDM r COO N ~ MN
' ' '
M- 00ON O N ll7GOtD~fI~M Inf~M C >\M 1~M OCOQ7CDCM M f0~ r
~7 C'7O
_ rN O r(DO O rr O rO r Or N r r r Or M MO r r (~N Q N D
~ r r
- r~ ~ ~~ ~ r ~~ ~ ~r O r~ r ~ ~~ r O ~ ~~ ~ ~ NM N (OM
H O ~ N
fDO I O1~1~O NN O ON tYaDV f~tpN1f)ODNCO Nf r
O00(DCDN N InMCOO N1~O (Otnf~O ~ ~ f'O N CON 1~~ Nr r ~ O 00
f0
d rr InO~ (DI 1-'r ifrdpr fvr COr Tr (ntnr O NI~r r M~ N (Dr lf7
Ir
_C
V
U
a
N ~ c0~U Ud _ ._OSL E ~ E N...E C~ Y C E
r,.~~ InrM ~ N NN :~tnr r rr r NM ~ft!7r U)rM ~ M NM ~l7M (D f0
~
Q UU dI0D D D DD m DO D OD D D OO D OD O DO O D dD O d U U
ai
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c'C- ~ N~ E = Od C
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O7..~r,._ OO ~ 'XO _ U ~ ZO O O :~O CO (C~ ~ ~
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a. ' O tn L- . f9U E
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ar~a caE-~ m v 01Qf'~~Inca ~ E ca~~ ~ a~~ a .E.__a Za~cLo oC E
i ~ m cc m ~ - p
-
C'3ad ~Idi= Y tlllilu.IF-~m U mm m U tlJU'Y Zm (nCnC3(n= iZ= CnU = ~r
v
I
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rM tCl(O ~ n,M~ t~1~tnM O~Yt~O (OC (DtDO fD~cDN V'etQ7tnO ~ I~
I
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m ~ 1~~ r
MN 1~O ~ _ f~t~O tn~ r m1~M InOM O OI~(OInODInt~Op~ M m
I ~ ' I OCD N~ N
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Q1 ON N Q (Dd0' O
N Or (DN O N NM ~ ~~ CO1~>> > >> > >> > X X( X O 4 0
I I C ( ~
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X ~I~ J JIJJ J JJ fnn n
-50-
CA 02290738 1999-11-19
WO 98/53103 PCT/US98/10561
TABLE 2 (CONT)
~ Cry N NO (D ~ COdue' N N N fD
C rf ~ tn(O(DtnInr rO r O N!7 (DO GOtt~~ r M r
O r N O '~rr r r ON O M ~'1r (ON O IQ'tnr O'a'
N N
O M ~ O fDr 00~ OT N ~'Is~~ N M I~~ ~-r~ IsOD~ r-(O
M n
CO (OV N CM ~t(D(OfD lf7N NO ~ N NI~I~LnM r (pN tll~
' f U7O (ON O ~f CO
O r N O M NlI)N 1~~(DM O ~ OM D ~ rN tf7COr M '~1'~ 1~N
d r N r 'cYrCOr llfN00~f7N ~ rr r tn
41
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M aom ~mno o ~rr~oo~raoNM a~o ~M ~ No M ~ o ~~
o r M V'OO M O Ind0(D(Or ~r M ~ N O N O ~.~
LnO O O ODO O ~ (DMO N N ~QfO ~ ~ ' O
O
N O N aD~ O NN
C r O tnIn(OCDO M COCOM r (DCOO I~h NO ~ r''O O ' Mr
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d ~ fDO ~ ~ X X X ~N J J ~J ~ ~X -~O J ~
-51-
CA 02290738 1999-11-19
WO 98/53103 PCT/US98/10561
C.'uncer Array
In the cancer arrays of the subject invention, the polynucleotide probe
compositions
on the array correspond to those genes which are associated, e.g. play a role
in, cellular
proliferative diseases, particularly cancer, where human genes are of
particular interest in
many embodiments. Types of genes that are typically represented on a cancer
array of the
subject invention include: oncogenes, tumor suppressors, cell cycle
regulators, genome
plasticity genes, apoptosis genes, ceEl differentiation genes, regulators of
tumor host
interaction and metastasis, such as extracellular matrix proteins, cell
adhesion receptors,
molecules that control cell invasion and motility, and genes associated with
angiogenesis.
In certain embodiments, of particular interest is an array having the
following types
of genes represented on its surface: cell cycle/growth regulators: apoptosis;
growth
factors/cytokines; oncogenes/tumor suppressors; cell adhesion, motility and
invasion;
invasion regulators; GTP ases and their regulators; cadherins; intermediate
filament markers;
receptors; cell fate/development regulators; DNA damage/response/repair/
recombination;
and angiogenesis regulators. In a specific cancer array of interest, the spots
are as listed in
Table 3.
The cancer array finds use in a variety of applications, including: monitoring
cellular
responses to therapeutic compounds; comparing expression profiles of tumors at
different
developmental stages; developing diagnostic tools for distinguishing closely
related tumors;
and the like.
In the following Table 3, as well as preceding Tables 1 and 2, the "position"
coordinate refers to the actual nucleotide residues of the listed gene that
are represented on
the array.
-52-
CA 02290738 1999-11-19
WO 98/53103 PCT/US98/lOSGl
TABLE 3
O C DN c D ~' OD cp~fV'
O t nN M O ~ ~ rN COOD
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t(7 V' rN N n rO r N r~ N ~1~MQ
1
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dp ~ O0 ( 0 '
C c h
f
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t t c0 vtY CDN M Q'r~ f~r ~ftD In1~.t~tDst~ C9
st
G. CD 1~ _h CD r Q1 f~ In rN M r ~-l!)Nr O~nM ~ CADOcDMM tn
I r NM ~ M 00 I
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-53-
CA 02290738 1999-11-19
WO 98/53103 PCT/US98/10561
TAE3LE 3 (coN~r)
0 ~ ~ N ~ fD CMD O O c~ ~ ~! V V' h Q~7 C~D
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~ w
CA 02290738 1999-11-19
WO 98/53103 PCT/US98/10561
TABLE 3 (cwN~r)
M 01M Ol CD M Of N
d' M O f GOtn ~COIn l0M O O In00V'CCO
~
O I tD O 'V'1~ _ M ~c01~a0M1~NCOGDM t~M NM l(~0)Y QOD
r O N O r ef N r rM ON InO Nr rO rGOrI~ r r rr
N
r .- r ~ 00 r p u7~ '"~prCDC)'-~.;~c0O'Of~tn r ~,r ~r
O nU N C
tn N OD O N t~OI~~f~7GDt7 OQf~ Vt COOCOOr-
O O rInN(DQfO~V'M cDtn1ON O~ ~ ht0.-tD
tf1
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Q
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aa aa aa a aa aa
Q a a a a Q a a aa aa aa aa
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f1
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d0 N ~p CO CO~ Or ~~ COO Vr ~pO ~f~j7N ~f~ ON
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d ~ ~ M N ~ M 1'~'d'InOr r rODr1LNN COOO Nr
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p
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U ~YmZ C c - u~~n r~c ~cn a i ic .
n~ nr.H .7
_jJ_
CA 02290738 1999-11-19
WO 98/53103 PCT/US98/10561
TABLE 3 ~coN-r)
r v o n [n r~n co
O COn ~fO Q) _ N tn V V O V' C~ OC~
trp n 01 NO M N n r V N r ~N OJ rr ctr
'N~ r Q COr 00 ~N O r r ~ _~ O~ M ~ .ij.p'nC~
~
Q CO N ~ lOM CO COV' CD O M tW l707OtD
r If7O 117fn C~ N O N m O CDc'~OD n rr (pO
N CDN ~M Cn C'7n n a0 N r cDQ7 cD N rr ~r
t0
1.
O
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U
~L Y -E C c0 L U ~ U L Y_ EC
~-io inco cnco co n n n n n r~ nn i~ n nn nn
a s a a sa a a a a a a a sa a a aa aa
'aN~
o
N N N
c0
X J J
p O ~ CD r In
QI N
k ~ O p ~ ~ (pD N
cD
CO n N ~ r N N O r
U X ~ ~ 7 J ~
G~
(n0 ~ iO fnON N I~NN p ~ (MOCD ~COO~~r ~ NN ~Cn
O
O r M~ r l~ M ~71~ O O CO~''~N N (D tc~NN _
'
O O p ODN r CD p 00 O O nr O O N N C7d COO
O N N Q~ m M N OM I17In
C~00 N ~ nIn ~ ~ ~ O ~ ~ ~ (p~ O O ~ V X~ X~
- X J J
-
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n m
r ~ ~ N ~ Z
U U U V Y U ~ Z
V
a~ YT Y YY ~~~ ~ YY
NZ _ _ m ~ U a Q UU Y + Z Y UU
v o u1
~ ui r c _
Yr H r Y U Z U
Y
~ Y Z a Y Y Y Y Z Y YY a ~ YY
Y Q - Q f- Nj
~ cp
F
a Y
' ~ Q _ ~ ~ ~ O Z
1
Z ~ Y
Z ZZ Z Z Z Z Z ZZ ZZ
1
1
- Q QQ Q Q Q Q
E ~ V Q O J QQ ~ ~ U ~ QQ
OC CL~ ~ a CL~ Y u ~fL
U l
ul
Y ~ Y Y Y .
Y
w Y Yr Y Y Y U ~ YY _~' ~j p YY
0 Oo~Q o
~
r ~ ~ ~~ U Y h- U
~ -
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(/J CCO ~ ~~ l- ~ CO CO m r N N ~tn CO ~~ CD nW
~ ~ UJ ~
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U
co
a~ U U UU ~ U U U U z Z U ~j = _-
,.. Y a
~ Z ~
O o u-- - -- - - - - - - _ -- Z W ulul
' J _ ~ J c uJ t ul tt~lu ttlul - + cD
t'-' Lu l ~' r' ttl
E-' W-
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t--a a aM a a a ~ ~ - a
a a z z
a Y a
~ ~ a ~- rZ ~ . ~-r~ r . mrw ~, ,.=>-r
~ I_r ~n ",~~- H I_ I- u F--F- ~-~.ao~ ~~
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_ 'D0 I- o Y N Y a fL Q z
V ~ ~ Q Y ~ Y Y lL ~
r ~ w Y
Z Z ZZ ~ Z Z_ Z Z Z Z Z ZZ Z Z ZZ
V + ~ - O U -O u~ Y - -- F-Z
Y ~ ~ f- I- ~ ~ I-- .-. tt1HF-
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r ,. r r r Y V Y U + Y
-SG-
CA 02290738 1999-11-19
WO 98/53103 PCT/US98/105G1
TABLE 3 lwoN~r)
C O ~M O ~fD cODN V
C Mc'n~GOM n O (D O7O O n ~ MN Mc0n t_OQ_~n~ N c_70
M V'c0 O
t f7M nO n M N r et T ~~p~ ~~ ~I\ V'
~y rv o~ N r ~ nco ~ is c0 r
Q nN ~r r W n n. co,_=r.M d~ oaov-_=~ aovo o v
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G. I Or NN 0) CD O r QIn M M M r00ODN CD r r~r O
nM Nr f0
d
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L
U
a
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L . r N NN N N NN
1 mm _m m r- m; r rm m m m mm mm m mm mm m m
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~ ~ a cn og ~- ' ~n
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m Z . D ~ ~,-' ~ a ~~N~ O
a u. ~ a U
d u1 ~ m g ~ ~ ~ ~~ o ~ 'm $~ ac W ~o V Q
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m
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_ cn (no~ gU f-YOI_OZO '7,~ EN UL IL<Ch- .n
v O ' V
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o u, ~~ zm =g ~ma ~ _~ ~ ~~ '~ z ~~ ~~ cc ~
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=
l0 ~ U () N N aN y d (C O XN U. lLLLlL _ fn
r ~_ = LL = ~ O r
I 'U
O ~ O ~ ~ J J QJ c N c0 u-~ ~~ a aa aE ~ Z
~ J lU d U' IU ~1 tn Zo c~CL cLm cL t Z
~ ~ tt~ U m ~ n a
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a Uv a Z U U U Qm ~ UU ~, F-~ OQ lh-Zi-II-h-~ U Z
a m L ~ ~ v ._~o ~
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U
-57-
CA 02290738 1999-11-19
WO 98/53103 PCT/US98/10561
'I~AHI.E 3 ~coNr?
c~ N ~ ~ M 1~ p r
r c~07 t0 N ~ ~ ~ t~ ~ c00 v a00 f00 M
N N r ~f tn N N O r r In N_ N N
CD O tn ' r (p
(D O ~ O ~ ~ O C~ 1~ ~ N In (O ~ (D OJ
c0 O 00 Q7 O In O cD 07 O C'~ f~ 00 N U7 07
G. N N N N In m N I~ ('~ ~ O N CO r
d
rr
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i Y
m m m m m m m m CO m m m CO CD m m
N (ND N
O t~ M h
O
X X Q °'
X
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t00~~ ~M N N c~ c00~ tOD~lOl~ i~ ~ N
C~ ~ ~ > > > > > > > > 7 X ~ X a X > > ~ X
m ~ LL1 w = .... W ~tj a~
a N (n at U r U U c
m ac°"n U" w ~ _~ _~ ~ ~ Q
Q V ul V ul U Lau V V V Q ~ V Q -~ U wi .° Y
U = D W ~ .-. Q ~ !- ~ e? 0 ~ D U 0 ~ ~ U p <° v o a.
~, ~ Y a ~ w H N m a U a c~ a p a C~ ~ p a U ~ ~m o
aQ Z p~- O~ a O~ 00--~ 00--~- O~U
w O m U a n. -~ ~ n. a. J ~ a a.
x ~ anm. aU H- acwn a~cwna a~cwnaO. ac~unm v,
Z_ ~ ~a H~ ~ Y~ Y_O~Q oYO~a oYtallOo'N N
Ll! Z z ~ ~ ~ 4' J I- J = f- ~N J = ~ ~~ J 1- Cn r 'C U
O, ~ ~ N O a l1J ~ llJ M ~ ~ tt7 LLJ M ~ ~ tf7 Ll! ~ ~ ~ -~ lL
cn .,. N m . a U U D ~ a 2 U D ~ a Z U ~ U a r
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7 n. U a ~ o Q r cn ~ ~ ~ ~ ~ L ~ ~ U ~ U tUl1 O U til V uOvJ a ~ U H H
!n Z ~ t ~ ~ U ~ a ~ d a ~ ~ ~ co ~ Q V ~ a ~ Q U ~ n- ~ c~ Q uJ ~
O _ ~ ~ °~ tz ~ U cn w O O a O >- a O r a O a a cn a n.
.C a a m a o ~ ~ O a ~ V p ~ ~ ~ g ~ 1 ~ U U ~ I- ~ U U ~ g U ~ O N
Z J_ Z m U ~ m ~ o > > m O ~ m O ~ I- I- m ut
d ~ Y U ~ ~ + a U ,v~ U V U O (~ ~ 'n V O O O 'n V U O O, Q Q
m v I-- ~ z m .. w + w v ~ ~ O ~ = m ~ p ~ z Ili ~ f- ~ _ c _
C' ti ~ a ~ ~ w L a v ~ ~ ~ ~ J a U CC J a U ~ a. p U 'd U
cv m I- m w a ~ a o~ Wn O O ~ ~ ~n O O ~ a- ca O U O o O
m= w~ ,,,,~ ma.0 ~E-cnw~~~cu ~w~aml°°uWn.uWn°- cn cn
ca m ~- - p ~- c - ~- cn m O a cn w ul cn m cn O a cniumn O a cn m cn a ~- cn
cn
~. O ~ ,L O o .~ z a Z ~ E m a- a u~ ~ u~ a cn a = a cn a = a cn a m O a c a
U~~ Ym e, x., aaa oaml.ua oaw awo~w~a~uo,ywau~Ya
.m ~-NZa-o o~ cna NaHa al-Nat- H-m-- I-aH-~ H.'f=--
~ = Z a ~ Y ~ o m Q O ~ ~°n ~ U O cn °_ cn O ~ cn O ~ U O c~ O ~
U O cn O O a = a
UZ~aXJ~' UV~VQrc~V=aUNUa~Ud~~aUa~.~-u-n~.UaVaD vD
-58-
CA 02290738 1999-11-19
WO 98/53103 I'CT/US98/10561
TABLE 3 ~rc>Nr~
O ~ O r
OMS~ ( ~ON P M M ~f 000D ~Q~
C
(p Oh.NU'1M MM d'r OM ~ ~O N NN O N00 ODr M00 CO
r Nr n07N rn ~d'N~ hN NN ~'N N~DMr f~(O NM Nr Lf7
I I!
I n~f7 ~-OD?nn M0 r~ ~r Mr rM I1V ppM rr 00
~ t ~ ~t I f If ~ C'~~ Ot O CD~fCOM r
~ ' 1C ~
t n D07O O f 0f Mr~ In CDN nC fD tD C700~M Q'~01N I nofO07 N
< ~ en wnM NCO t 0r f~O ~OD M 1
mI I O h t
I f
G.~ ~~ ~ rr NMN rV .-h MV'M~ OM NM r~ wM NM NN rOD tn
( I
d
Y
_C
L
U
a
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' ' ~~ ~~. ~t0 ~V'~tntn1n1f7tf7tnin1n. tf7U7Intn InCO(O(D c0
V tn
" M MC V eT V mm mmm mm mm mm mm mm mm mm mm mm mm mm m
Q m mom m m m
cD
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CO Q~
M
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n rn
c'm ~ ~ ~ N
rJ
as o ~ ~ Q g Y
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1~.COr NtnInd0InOOf~ ~fGMCDCOInMM M_ MM OM MO Nr N
m N ~ p 0ODh1~N1~rM 00CDOO OM OO p'~ CDIn00M N
~ O
L'f~O~ M O r (DCOO 0M rr NO MO NN rr rr rr ~1 (pfDrN
U7 ~1 ~DrrN (O pO 0p ~N Mn 0O ~ pO M O~ O O
M pNM
~O a0O) ~ ~~ lGt f~7O~lt 1t G ~ 1.L llllL N~ xY xI~
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-59-
CA 02290738 1999-11-19
WO 98/53103 PCT/US98/10561
TABLE 3 crc~Nr,
__
C~ c~ ~ N o n cn o
OI~ ONOON M V~") f~00n 00 00
O d' tt !Y r N Ch r- C~ r tt (O O (O ~ In fp C~7 tn f~ C' f~
N ~ O N c0 ~ ~ N CD O N n N ~ N Pl N ~ 'Q ~ ~ M '-
r CO r- ~ In CY1 r l0 ~ r ~ ~ r In ~ N In T ll7
N N O O O) O tn n r ~ M O GO M M N O M ~ CO O r
et O tn f~ QI O V' N O N In O C'7 n N CO M ~D O1 ~1 r M
G. ~~ tnC~OfcOr.- O~cOOCONr- NINNY M C c0 CO
Q1
wr
t0
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O
O
U
L N L ~C E C c9 L U ~ d Os L Y E C
Q mgt m m m m m m m m m m m m m m m m m m m
c~ ~ c ~ N a~
co
'ri ~ y i ~ n
C
ao v cn ao o r. ~ v N ~ r~ _~ o ,. o n o o ~ o
m v n ~ °° ~ ~ ~ N n .- a> co c~ c~ o .- a~ ao o ~ o
C O n ~ O O ~ LO N ~t ~- N ~ tn O p N c0 f0
O ~ C ~t N ~ cn CO N O ~- ef ~t (D ~ M ~_ ~ ~ 0 ~ ~ O~ ~ tt
C~ > > ~ ~ ~ > > N a ~ X X ~ X ~ ~ D ~ ~ ~ ~ ~ >
a rcn N
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a w ~ O Z ~ O Y a 0
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N O O
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x x ~ ~ Q O ~ ~ '° o N Z U ~ O O O ~ Z
m Z a. X ~ p a ~ ~ ~ ul ~ o Q U Q U Q
H C'3 ~ 0 ~c%~ c.cm d mQ ~ >a > z D
,0.. LU Lll p V ~ 1"' ~ ~ °~ ~ 111 ~ CO ~ O Z m ~ O U Z
~ ~ ~ Z fn O a O ~ -p ~ ~ Z U ~ m O Y a ~ n
00 ~oUN c d ulaoCul u.la-a0 a ~ p
m co ._ ~ cn .-. ~ cn cn o E- 0 0
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Z
a ~ a m ~ N ~ ~ ~ Y Q LU Y Y ~ L~1J ~ L~1J W
a~ Z -- C~ Z Z
~w°i~ ul Hu°yU~- E EZN NM ~LLOu~ wo~a >
~ CL Z ~ ~ ... c UJ U ~ O c~ fL 1- F-
O - ~ o o H ~ D ft .- D D
~ x x ~ m w a z N :n - N ~, c9 w c '~ m O a n. O O a cn N 1- cn cn
C~ ~ ~ C O z O ul o ~ m ~ m O oC .~ E ~ a Y ~ a ., a o Q a H Q ~ Q
N a a ~ U a cn Q ~ ~ o o Z O o o .-. Li.l ul ~ lil ~ u! ~ m , ~ ~ m ~ m
~. ~_ c~_n ~ O U Q m + Y o o ~ ~ O ~ ~ ~ Z Z U Z m Z a ~- m ~- v7 ~ ap
U z ~~am E~v~~' ~'o~ a ~o cncnmcnacnUQama a~
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~ ~. y--
V ODt~J7aSC~"'mm~~~n2~~~aa ma~tn~~~'t a~acnaafO~'cnN
-60-
CA 02290738 1999-11-19
WO 98/53103 PCT/US98/1OS61
TABLE 3 (cwN r)
O ~ C7 Q V O V M In M ID M tD Qf OD
O W M m f~ ~D CO h M M O M O 00 CD N M M
CD r O O 1~. aD t~~ O O 'a' X17 f~ h 00 C' op ~l7 (O
N p~ n r N M N M Q In r r N N N r r r
(fl O CC O O O ~ O h r /~ r OD GD 07 M O t0
1n M 47 N ~' ~ (D 00 N r Iv N m tn O N N 7~
N 01 h f~ M N et t~ 1~ c0 tn M st r ~ ~ M
O. N CO tD r N (D N N M tn CO r N N N
r
(0
a
L
O
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wa v ~ m.... o~ s._., ~_c_ E c m a v w
L r r r r r r r r r r r r N N N N
Q U U U U U U U U U U U U U U U U U U
m
° ~ ~ rn
j_ ~ = X
C
uj ,_ l17 OD V' O r In O ~t ~. O CD O r O CD
C~pr'OOD OMf ~~ ~ n~00D .M-NDOOD N 00
d ~ (wj~C'N~ f0~ r0~0 OMD OCMOM r~~M ~ MO
C9 ° °~~ ~ ~x x x°o °-~-~~ x
Z
C4
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a. n m x m _ Z U w m y ~ ~ ~ Z w ° w o
D U a Z m n=. Z ° a m ~aC o x n. > x
~ Q ~ Q Z ~ a = a ~ j Z
ZX v°~ ~°mm F- z C ~~U(n Jw -~w
O v ZtLUUZu.OJ N ~ c--~u VOa~ VO ~ VO
t- + Y d > O o U ~ O m U w _ pC ~ p CC aC
a Q m H ~' cc m H U ° w ° ~a m a ~ ao o a c7 a
u' N ? Y x ~ ? U Z ~ Q ~ w X Z Q = X Z X Z
Y ~ ~ V Y ~ V V ~ V O O N Z Z m ~ Z Z Z Z
~ ~ Z O ~ ° ~ U °° O U ~ ~ Z O O ~ Z ~ ~ m Z ~ cv Z ~ ~n
m w ° aC = Y = Y ~" w Z O ~ ~ w ~ U ~ w ~ U w ~ U
~QZ~ °~°izum~~~m ~m ~ _ = aw~Q~ w~~ w~~
e0 0- _ ~ w Q ... ~ U Q O U m p r N w m w w
w Y U E- f- U p ~ t- U co H- f-- ~. >- ,n ,~ o ,n ~ a O a O ~j a O
~ Z Q Q J r~ Z U ~ ao 1- U O x x ° D o ° a ~ U Z ~ U Z U ~ U Z
~ u7 ~ Q U w °- a w a Z m O O 't a a E Q _ _ O m uW O ° w oC O
c9 w
~Zt'Q m~=w~~_=Z~~ rto ° ammtmwwV°-p= Ua~xUaO
a ~ z ~ ww~nmaQw~n.° w~aww0~
rOaocU Q~a~=~z~» ao ~ ~~Z°Ha~oc ~ ~ ~-~ ,-c~
~ a U Q O O Q m ° O Q m n. O
w z m ~ m = w a ~ O O Q O w w O c,9 ~ O c9 ~ O O c9 ~
O w ~ w ~ a ~ cO ~' a ~ U ~ a' Q ° CC 4 CC CL Q
L Q Z tn C7 C~ Z ~ Z ~ ~ ~ CL 2 ~ ~ ~ ~ a Y
wQ z>-wzaowza°~z= > ~aa -n. a a a. a.n.a a
C~m°z ~°w°Y°w°YUp- w 00 °wa,
°m °w
w w ac ct o o~ cn cn cc cn a~ cn ac cr cn
Y ,ga~~?''~,.z,.,~-' o~~ncn cn ~aa ~a00a0°~N
a0°~aa0°~
~~az E-a~Z~~aZ°~xUa Q ~aa Eaa.aaf-z af-z~n.f--z
4ww Qo~mQO~wQO~ x~ ~? OwwLwppw~0~ w'zOmw~zO
U_ ° p I- - G. p O ~ 1- p O cc ~ p w J M J v a ~ aC LL 1- I- ~ U7 ~ ~ ~
(n ~ ~ ~ cn
- Q Q O ~ m a ~- r U a I- ~" U °° Q a '~ a "'' a a 4 't Q Q a a
~ U U Q U a a U
~ Z Z ct E- Y f- ° = Q ~- ° = Q ° Z Z Z Z Z Z Z Z ~ Z Z Z
Z ~ x Q Z ~ x Z Z ~ x
U D c~ a ~Q u. Q Q ~ ~ Q a ~ w Y ° ° ° ° °
° ° p m ° D ° ° d w u. ° a w
° ° a. w
I_
CA 02290738 1999-11-19
WO 98/53103 PCT/US98/10561
TABLE 3 ,cc>NV,
C ,~ o ao m a~n rM
I N N r CO M fD O CO~ V'O V7
r
Q W (D ~ O Otf7 N CD CD CDn c~ N OIN_ _ NV M
D ' N N 00O ~(7f0 N r I1~M M N InN O?C rr N
O
t
CO a0 P m ~ r~r ~ n r ~ vo v ~ ~r .~c no r
n oo m a
N O (D ~ M (DOCO OD N O CO'7In tn O Mh rCDNO O
0 ' 0O OD M m ~t CD ~ n n~ OCOOO n
' O~
c
O.O N ct O n cn O V a0 r r17T M N MCDMM O
v u7 N N .-V' X
I
r
t0
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a
N pf t ._..~Y N E C fC tU ~ N M Mt ._. YM E
N N N M MM 0'7M M MM m M
Q N U N N N NU U U U U UU U U U UU UU UIU U
U U U U U
co
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m
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0
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C~cn C ~ x ~ ~J ~ ~ ~ ~ _~ ~ o ~ ~~ oa a> >
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-62-
CA 02290738 1999-11-19
WO 98/53103 PCT/US98/10561
TABLE 3 (c'c>NT~
r ~ O~7tnO Oft(7 Of_
r ~ r N~ ~ N VO'M tn ' M
~
_ ~ ~Inf0~
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N ~ C~~ r O r r ~ ~ m N r O C~ N r ~O CVO
In rV'tDCO_'00 r r~ '~ I~O ' CO ' ~t ' In r
OD r'~p r M O inV ~ O mM OM .a.M fD(O~ O
0 M 07MV'OOIOO (Otnr O ~!'~ ('7CAD'V'O Q7r Np ~N ~r~QItnCDODr O
~ cDr OV' O C'~O~v aDN tt~et cntnCDv
cD O OcCc~c0vQ~ c~cDc V r C~lI71 N r C7 rN C~r GD(~7NM r(~r1p
tt~ ~prr fhInrtn O ODr r
m
f.
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CA 02290738 1999-11-19
WO 98/53103 PCT/US98/10561
~I~AE3LE 3 ~corv r)
C ~ n N M ef N aD ~ n M 00 M
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-(4-
CA 02290738 1999-11-19
WO 98/53103 PCT/US98/10561
TABLE 3 (coN~r)
O D YD ~ OfY07DO N 0 0sff7
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-65-
CA 02290738 1999-11-19
WO 98/53103 PCT/US98/10561
TABLE 3 ccoN r>
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-66-
CA 02290738 1999-11-19
WO 98/53103 PCT/US98/10561
TABLE 3 (c'c>Nr)
p r O W M_ M N U7 C~ f0
CDC Q1 ~N WN ~tM tn O c0 M CO
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_V I -
CA 02290738 1999-11-19
WO 98/53103 PCT/US98/10561
TABLE 3 (coN~r)
Q ~ 1~ M~ ~Q~ O ~ ~ Q0~ ~ CDO 1l!
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-G8-
CA 02290738 1999-11-19
WO 98/53103 PCT/US98/10561
TAt3LE 3 n'oN~r)
h N N M N N ~f7
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-G9-
CA 02290738 1999-11-19
WO 98/53103 PCT/US98/10561
TABLE 3 U'<>Nrl
C t fiO M 0 _
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1 N MD (D t ~ tn O
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w
CA 02290738 1999-11-19
WO 98/53103 PCT/US98/10561
TABLE 3 ~c~orrr~
_ ~I7 O ch I~ cD ~t7 M N tD CD ~ GD CO OD tn ~I7 Of
et CO r N O CD N GO tf7 M Cp ~' f~ tn N tn
~ Q M O C'~ CnD fD tn Q M c~ f0 c0 M m 1~ O r dD tn O r c0 N
r N r N O ~7 N N N N ~ C~ O r r Is tI1 CO C~ N r fD r O C'~
Ul N I~ 01 ~ 00 ~ r 'V p ~p r O N O_ ~ CD CD h M O O CO r r r r r
O r t0 f~ n ~ OD ~ O CO C7 00 V' r CD GO OD 1~ ~ O N V~ tn
tD r O I~ ~ V N tn N O O) N CAD N O Q~ tn fD OD 1A f~ O O N t~
r N r r t0 07 N tn N N N 1~ N ef r r (D t(7 ~ N N V (~ GO M
d
fr
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a
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LZU Z ~ w z Q Q Z ~ O ~ o, a Q c ° = ~ Y Z CC ~
d Q ~ w g = ~ U U Q t11 ~ a ~ a ° d N v O ~ ~ O
LU h ~ - d O fn J Q (n J
c .C D E ~ CL
O Z ~ Qv O m V '' = O Z Z_ O -~ ~ m J '~-O r G7 N ~ tUl1 0 twit 0
a. ~ ~ m f"' ~ ,t Z z z Z d ~p~ L v v j ~ ~ ~ Z r . d Z
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Z Z Z Z Z Z Z Z ~y,~ Z - Q Q ~-- ~ N O d O t s ~ c d 'y 'y - - 2 Q J Z Z Q -~
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d OQQIaQaQQQaQ=QVaQ~~~a y ~ m ~ ° ~ c5 m ~ d ~~n-zoCCCaZ
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~° ~° O ~ Y a N. t,u Y
_71 _
CA 02290738 1999-11-19
WO 98153103 PCT/US98/10561
TABLE 3 (CONT)
C' N V N
CO 00 CO O Q1 t(7N 1~ 1nO C~700 MtD 00
C 00 OI N O CD N ~ a0 1~ (OC'7N O07 1~ 1~r N~
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0 h N tn Q~ ~' a0 ~ (D ~' r NO f70tnN i~ Inr M1~
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d.O ~ N N (D Q7 ~f 1~ r U7e-OC~r h,f~ Inrr Or
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7_
CA 02290738 1999-11-19
WO 98/53103 PCT/US98/10561
T~A(3LE 3lcc)rvr)
N~VM~~0~0 O V c0
M (D r OD 1~ ~ O CO ~ CD O OD (D ~ (D O O V
r M r N ~ r V' CV 1~ r r- r ~ O ~ ~ O ~ fOD ~ V lI7
fD r ~t ' In ~ OD
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N r ef r f~ M ~ M 00 O CAD N N r 1'. Q O O U7
d. r ~ r CO N r et ~- M r r (p N U7 M r r N N r r Q
d
~r
f_.
1
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ea
E C f0 d U ~ 47 O) L Y E C c0 L U 'O N Q1 L
r r N N N N N N N N N N N N N N M M M M M M M M
LL LL LL lL ll LL I1 lL lL Il ll lL I1 Il Il lL lL I1 lL LL !1 11 lL LL
fD
r
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O c~n Z .:.
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Q ~~.~.~o a~~=
c~ ~ c~ Q
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tiJ C~ CC CL O ~- m ~ p ~ uJ
uZJ LZiJ m ti a ~ LUtJ O O U CC m m O ~ O
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Lll LU LU Z LU V- ~ 1_ O O = O ~ O U' Z ~ ~ O ~ Q J O
Z Z Z Q O x O m J Z CL ~ O ~ ~ U O Z m > ~ Z -~ = C~ ° O
o~~~ m~ ~ zg~mzQ°~°~gg °z°~~ ~° °-
~wc~ nm
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-73-
CA 02290738 1999-11-19
WO 98/53103 PCT/US98/10561
TABLE 3 (cc)N r)
u~ o M co
(O r n r m ~ N n CO
n n o m o»n cmn a~ a~ co v o co co a> m n a> ao o
O m r N N r N M O ~ M
. ~ ~ ~ ~,N,'~ (ND ~ ~ r <D O r ~ ~ r ~ f0 st N U7 ~ M fD V O
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M N M r V' r fp
G. (00 ~ ~M r000 ~CpOCONNrO~r
d
l0
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C7 C~ M f~ M M ~ ? ~ V' ~ ~f V' ~f ~t Q C' V' et V' u7 In 1I7
_
N
N
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~f er 'ct r ~ N
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co o ° N ~ v ~ ~ ~ ° ~ v ~ ~ o ~ ~ ~ n N
00 ~- ~ n O N N ~ O In aD n 00 n p O ~ r ~ n n tn
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d N CO n r O Q n N N (D N p p r O O p
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Z Z U ~ m z ° O O ~ O m m O w cn ~ U
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v a
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w = d z N w ~ ~ Z = V Z ~ ~ U w O cn m
Y O~ m w ~ IL ~ ~O H ~ J ° = L
C~ 1- c%~ ~ U a = Z m d ~ = c
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t w U .-. Z w d ~ ~. d d Z O O U U d w w 1- C~ O w ° r
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d a W ~ CD O a ~ H' O F" - > ? ~ d Y Y Y -Y Y Y z ~ M Y Y U Y ~ Y Y Y Y Y
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J J J ~y J J J J J
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c~ IJmQwmQm--w.Jww~c~oJa ~~zzzzzz~~~z_z~zNzzzzz
U~a.d~==w~a= ==~=Udu. - - ---
CA 02290738 1999-11-19
WO 98/53103 PCT/US98/10561
TABLE 3 lcc~rv~r>
_.
p c'~ O cD N aD M h M !~ O_~
CO M U7 1tf a0 O cD O N (D u_7 <_D t~ N ~ ~ ~?' cfl V ~ ~ m p~f N N O N
r M Iw O M OD In N I~ O M In N N r N O r
N m f~ ' (D lf~ ~M. ~ In lf7 O' V ~ ~ r r p ~ r ~ r lf7 ~', r ~ In O' (D r r
Q N In OD CO fs ~ N r (O P N ~ N f0 fD In 00 C~ O ~ OD O1 M Is C~
N CO .- M tn O ~ 07 h OD ~ c0 N ~ N c0 V' O Qf I~ ~ ~ M M M N (C
a (O N r M N O M M M r N f0 h N ~ r M CD N O M N N (O CC
Q1
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L 'O p~ Z ~C E C (G .0 U ~ N L
~ t~7 tn tn tW f7 In u7 tn ~ ~ (O t0 CO ~D CO c0 f0 CO CO CO fD CD CO CD f~ ~
1~ ~ h
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m 01 r ~ O tl) M N M O f0 M r tn N N Q f~ r ~ M In f0 N In ~ ~ ~ N r O lA r
N O ~ et (O O O O ~' Q~ O O (D N tn I~ M cD ~ O ~ O cD 07 O N O N O O P ~
C M CO M ~, N N 00 r N M ~ M M p r ~' (O N N (p (O O r O f~ M (O M N CO O 1~
d O CD l_lY r M O N O O r N O ~ 00 et d' O O '~ M to N O
C'T ~ ~ J > > ~ ~ X X X ~ a X ~ ~ ~ X X ~ J ~ ~ ~ ~ J ~ J Y X ~ ~ J
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~, w w vw w w U U U U i.-. Z Z m -~ -~ m " U m O f- ~' w ~ ~ can
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VIZZ,Z~ZZ JJJJJ~ZZ~Z aan. cncnnnmH-.r...-..r C7f-f-f-~nU~-f-UUU
-75-
CA 02290738 1999-11-19
WO 98/53103 PCT/US98/10561
TAE3LE 3 (cc)N'r)
M ~ I~ ~ N
O In ~ 1~ ~t Nh
N Q~ NI O NI~ C
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N
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I Z
-76-
CA 02290738 1999-11-19
WO 98/53103 PCT/US98/10561
Apoptosis Array
In the apoptosis array according to the subject invention, all of the unique
polynucleotide probe compositions correspond to genes that are associated with
apoptosis,
e.g. cell cycle genes. In a specific apoptosis array of interest, the spots
are as provided in
Table 4
_77_
CA 02290738 1999-11-19
WO 98/53103 PCT/US98/10561
TABLE 4
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d ,
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Y
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w
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a p
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~
a~ ZIZZlZY Z Za c ~M CCCCm ww ww wY ~ ~ p
YY Y Y Y l~ a ww w 1-I-I-1-h- ~
J YY~~ ~ a N UU U OO OO OZ ' c~Ua
p z- W
Oz Z ZZ ZI-Z Zw z > ~~ ~~ ~w ~ju.cC
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~ ~ - - -
~
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J J J,JJ J J U I-J NN N Z~Z J
~ J JJ J J p ~ ~ UU U YY IYI~~ ~~~~ U U UU UU U UU U
a a J U - >-
IJ
F-
d J m . m w~ >-U ra pp p JJ J wwuu w~w p p >->->->->-~> U
U mz IUlwlmtma U U U N UU U UU IUI~~I~ ~~I~ U U UU UU U UU
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t7. X~~ I
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_7g_
CA 02290738 1999-11-19
WO 98/53103 PCT/US98/1OS61
TABLE 4 (CONT)
i
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Q ~ ,w I ~ ~ yn I y wmn u~ ca ca cfl cn co ca cn co co ca i co
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r
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H
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n a~c'~'n ~w ~ a~wac"'na a a
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1- z ~QZ r4- pQdpYp p 'p
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z ~ Z Z ~ n U - p Q Q p p D.. p
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I- f-wJOJI4T'~rZ'(npr'N ''C~CD~ CC CLd~Q~Y
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-79-
CA 02290738 1999-11-19
WO 98/53103 PCT/US98110561
TABLE 4 (CONT)
t '
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N n JI ~I J ~ O ~ >'7 ~I~ ~IX XI(OifO XIX W-~OIJIO,~iJ
G.1' X
CA 02290738 1999-11-19
WO 98/53103 PCT/US98/10561
TADLE 4 (CONT)
V
C_
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-
CA 02290738 1999-11-19
WO 98/53103 PCT/US98/10561
TABLE 4 (CONT)
::
ca
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U I
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r ~ r- ~ I r I
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rlrl~rT Tr' fT 1- T
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Y c%> W w w O
w Yaao oC ~oC ccwn ~ cwnjc~n~cwn~cn~cwn O
U m oC O r Q O - Q N Q Q o >- ~ Q p >- ~ ~
u. O ~ X w ~ u.. ~ ~ = w w p U = ~ c~ U = ~- U
~Uy~ m ~ w v Q ~ ~ w ~ ~ ~ Um ~ 1- ~ ~ O H ~ ~ Q
w ~ can a c~ U °- ~ a n. ~ O~ ~ °- g 0 ~ ~
v C'3 C'3 ~ c Q U ~ c~ Q U U ~ U d a.
U m~0 D_ N V wcn ~Z w ~ r-~OcanE-~Ocn~ N
w O O ~ ~n m a Q U 0 O O ~ a Q 0 p Q U ~ ri
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n. m ~ O ~ f- Q ~ cv ~- O w Q a W v O ~ ~ ~ a l.Uvu
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w z ~ Z Z CC? 'o p !- m wi ~ N- V U U ~ U U U U U U
I- cn ~ w w c z tn m cu O Q _ ~ C7 w ~ U Y ~ ~ co m
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az~OY~~C'3 io CC ~Q ~''~ N NINO~f~NO~QN v
r~ lLl~ ~ ~ ~ m ~ 7 ' ~ LUl! U ~! _ ~ U.~ ~ ~ LaIJ ~ ~ 0 ~ ~ ~Iw O
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I
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N; NIOD N m,? ~
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_g7_
CA 02290738 1999-11-19
WO 98/53103 PCT/US98/10561
TABLE 4 (CONT)
d
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0
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Z O m U D w N
v r r N N N N N N N N N N N N r r r r
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m ~-a~i=-z za '' ~c_ia_~a_o '" Q_ o
w w O m 0. O Y O U '' U N m Y ~ m Z
n. O Q p ~ U c U
o J cnUm_Qw cnQ ,.; =O=Ox z c~ ~
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N
YZY~z ~w U O Otw- ~ w
f- u. pw a=a=O = U cn
Z ~ Z iw-. ~ U ~ U U rt = o. = n. m f-
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p w cnwUwa aN U z~~z u.ct wcn o
rL a °- Q m J ~- u. a, = w Q u~ ~- w N in z m g u~ a
a. m 2 Z ~ ~ ~ p ~ f... a ~- t-- z t- z O 'g y. p a
v o ~ ~Y~ Paz z~. o OwOwa m N,- ~o
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w Q w z Q O J Q u- U .D ~ Z
w U Z z ~p~. O O Oai--
vj Q U Z ~ Z ~ ~ + ~ U d Q CC ~ of ~ Q'~' Q~iY ~ Z ~ 2 2 a Q IN
ar IU yn c0 - U - Z ~ Q ~ w J X O p 1- m Z m ZIZ ~ d~~ d ~ ~ O
~~ Vt~ Q Q d a 4 CL fn a Q -~ a J X ~ 0 ~ = O = (!)iJ Y C'3~~ ~ _.~ Z Q
d ~, IzCCCCrLm~twQJwO_wQ~
U i~ II-1-II-1-YI-cOUQO~CCUDF- UO-Iz~Z~!X-~ JI-J~JH-u- Q
J
O O fND C~~j t~f7~ ~i
r f~ r O ~~ MI MI C7 O
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N SD C 01 h N ~ GO ~ Q5 I O V' O ~_- N Q7
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cal I~ > >~ ~!oml a > > a m
-83-
CA 02290738 1999-11-19
WO 98/53103 PCT/US98/10561
TABLE 4 (CONT)
V
I
I
I
d
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c
a
0
0
U
A D ul ti CJ Z m U D ttm~ C~
C'3 Z m ~ U D to u. C'J Z m U O ul u. C7 Z cn U
~- v Wn W WW Wn tD cD cD c0 O ca cD h 1~ 1~ n n t~ t~ m C7 W ( m O O
Q r ~ r ( r 1 r r r r r r r r r r r r T r r r r r 1~ T T r T r
O c
~c O
O O .c a v
U U ~ _ ~ r
c
Q
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0 ~ O i ~
r2 w o
U ul ~G ~ a>
U J ~ U io
a CL Z c ~ Z c~'o
Q ~ ~~' ~ ~'~'=Q
In ~ ~ D ~ Q (n .N N cn r
D ° CC = Z O U m_
Z Z ~ ~ M E ~ ~ f- O LU L m UJ
O w ~ r ~' mn m
OOp O ~ '_ ~Ua ~ n. ~Q ~ ac ui
yu ~ Z Z a a ~ -Y Q co
aaa a ~ ~Q-~~ Q a o z_ n~ ~ ~_a~
O O ~ ~ w a - - m = w M Y ~
H H ~ ~ U m O O O O U D Z Z .'~ ° r
~ H f- H Z N W ~ Y O
J m + Z ~ ut w ~ _~i _m a .t
N ~ E- f .c ~n
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m
c_~ c_n U U m D m I Z S I- -_ m a a a~ '° m -° o c s
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o U Z w Lu ~ ~ c o
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d a a ~ O ~ Q O ~ ~ 2 ~ U ~ X = Z ~ I- F- °' c ~ s c
c00~0_m0 C7~inC7C'JC7n~" ~~ f-- '~U U ~ ~~ a~ ~ ~o?~
C~ Q QIU r;U N U U fO Y Y Y D o ~ ~ ~ ~ O O ~ o o ~ ~~ ~ a
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VHF-,~E-I~F-~O ~ZZZZ,-UNMZ~~CO ([= CI=~V N ~-%- i
u1 LtJI ~I ~ I-Z t4.d~-n.O.~ o
UoCOC~OuuOmQO~JJJJO wf-~ E- movN ccO~ i
-UU,~Ui~Ucn1-aJ»~1-~~moomDmlnt~ =a ~Q ~Ur~- ~~-a ~E
alLtllu~tl!'~~LUQ>-ulmmmma plLli.u.ZtltLti ~w ou! ~O3"c~ ~Q~Z ~cd
Ucncnlf- -- - -CLIE--~~UD~ZIZZU v~~C'3C7~pC9UC'3 Op 'pu..n.aa v~(rD m~v
I i
ca
M
~i ~ ago N m ~ ua~'7 I
M ~ ~ M Q~
ye ~ I' ? ~ rL X ~ ' m I
y CO M i CO ~-" "' a0 ;
O ' ~ OMNN rV WY7~MNf7D'tl7M.tD
COf01 ~ ~_pflnNN~~~' tt~(_O~ ~O(DO NOf~000_O_MaO~oON;f~
~i N~~ M~~OD~M~OI~ NN~ '-NuflN CMD(OcDaO~'')I~c'N~ct~000N,c0
' 1~ Q) ~ O 1n tD P. (p M O ~ 11 I ~
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c7 a~a~ ~' ~~rm~a ~- ~ ~~x >-~o ~ ~ ~ ~ ~ ~ X x cn ~ ~,~~~~ma~~
-84-
CA 02290738 1999-11-19
WO 98/53103 PCT/US98/10561
TABLE 4 (CONT)
1
m I
..
~o
c
0
0
U
'~°ZmUm~w~C~mU OwwC7 m1U pu~u.c_~ m UOwwc~mUDwu.
~~OpOOIQ710710000 0000 rIr ~-rr N NNNNNC~C'~C~(")C~
Q rl.- r ~W r r N N N N N N N N N N N N N N N N N N N N N N N
N r
m ~ Q
m m Z Z
0
41
~n
C~
O D
p Q ~ Q Q
a p~ C3 C~
°' Z Z
m ~ ~ H
... O O
O m om,. a
H O J J
r p Q Q L m m
f' is
Z
wm a ~ CnU',~, U' ~wz zo I
r (L = Q ~ ~ p Q N
U p J ~- V r IV- ~ ~ O ~ r w
~ j Q Z Q ~ m N Q V
Z m m Z ~ s ~~O w w w w Q w Q U Q CL c X W a ~
r Q J ' ~ ._ o ~' Q Q Q Q ~ ~ p Z p U ~ CL m Z w
Naw oz_.~ om°wacc~ o~cL wwoawacn - ct~az
v a'~ c~w~-''~ZQ~u~.. u~..~ a~aDOOQ
c Q cwn ~ O c a W.- c4 cn cO c~ p Z p ~ p CC o a a = O
o.? '=u-V.°°~ E ~uZ',yQa QQ p~~Zaz~ p wu'~ct
~ Z Q .- p ai m 1- p CC ~ ~ ~ ~ ~° O Q ~ a ~ U uU.l uV,J Z a
Z ~ C Z ~ u- U Z a 7 O w f-; H 1- !- w fn ~ f.- m f- m
,y °' ~ 'a~ >- O Z ~ w E v~ ~ m cO cn cn cn a w = ~ U w U a p p p Q
d .-. ~ ~~ ~ N U ~ '° ii ~ '~ w w w w w w Z p m Z cC ~ p D Z U U m w
co -~~~ a;wQl- p cOZZZ ZZ 20 CLD~p >-
> a>W~~~p.. ZyZO00 00 10=~4ZQZ QQI-w-aQ~ft
_ai ~ E v ~IXiCL ~ tL o ~ 2 Z = = Z _~=Z H -U = w I ~ C'-3 CC? O V V z O
0.~~~OwCn V 1-~ OzQQQ QQ~QZ=~O~O JJ~
o ° a~CC U Q a p ? .~_ Q ~ ~ _ _ > >~> j O O ~ O ~ a a ov N Z I- I-IO U
L O ~II_.~ ~ ~ Q J ~Z J J J J J j J J a cc a o~ a n o Q w w ac X
U U ~I ~OIZ Z 1- Z D U map C3 C9 C7 V' C'3 ~n.C7 C7 Z O p O p Z Z U U tL ~
~IU. w
m
c_~
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j I
U_) I~ _ cD 1~ ~ c0 ~ N C ~ cD cD ~ N
CtD (p ~ O O 0 N ~I O ~ N N OOD ~'i0 mIcOD O O ~_ ~ O O O
m Q7 N N O N O O O Cfl 1~ ~ O st ~ I .~- M Q ~ O t_n tn ~ O ~ N
CNC~ItII~QfOMm~_ ~IN_~_M C0ln _NQ70 O _1~-COCD I~
N 00 f7 h N O ~ O O n O O f~- O~ ~ ~ O O ~ O O 10 O
C9 > > > oIJ ~ ~ p 01 ~Ip X ~ X XI ~;~ Xltn tn ~ > > J J X ~ XI~ J.
-85-
CA 02290738 1999-11-19
WO 98/53103 PCT/US98/10561
TABLE 4 (CONT)
N N
o ~
Z
J
N
cOY
N
~N
V
N
C ~ N
o O U
O N
V Z C '3m
NN
Q D U piJi'JZ - ~ -~ ~ pa
C tl C Y NN
r
r
N
r - r rr rr r r r
r'
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n
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C
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o
Z Z
w z - ~ ~ ~ ~p ~ w
O n z
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U
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Z . Q Z ~Z = >.N- O~ Z
d
_
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p O tiZ O Z Qa cn~ C'3~ ~~r Q Q opr
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d (~ ~ >X~Y~ X X
N
C9
p
CA 02290738 1999-11-19
WO 98/53103 1'CT/US98/10561
Human Stress Array
In the human stress array according to the subject invention. all of the
unique
poiynucleotide probe compositions correspond to genes that are associated with
stress
responses of human cells, e.g. stress response regulators and effectors. In a
specific human
stress array of interest, the spots are as provided in Table 5.
_87_
CA 02290738 1999-11-19
WO 98/53103 PCT/US98/1O5G1
TABLE S
Z Z
w w
p J m' c n ~ a
n
C ~ m > z Q ~ N Q
r = Y ~y u~ m
~ O Q ~
_N ~ Y o0
N can U Z Q Q ~ d Y Y
Z Z_ m
~
> Z O Y Y 'n Q Q N
c
c N O z Z Z
a Q Q C ~
a Z Q ~ ~ ~ v va C C ~n
a~ ..~ ~ w ~' w
a
- r-
d j~ ~ Y Y Y Y~ Z Z Q
~
"-
a W C C C C Z Z
m u
m
Q ~
~ ~ . '.i. w~ O ~ Y
~ u '-' ~
~ ~n
U n N .. ~ ~. .. ..~ ~ ~ ~
, . ~ ~ . i Y
~
_ _
~ N ~ Z
Q
Q. N ~ N N N N N
i
C ~ L" w w w w wQ s w
Q cud
U ~ _ M 'G ~ pw N N ~-
N Q
a
DC
!i v7 ~ ~ a ~ O
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_ t cn v7 pp
Q
.
V
W ~ ~ Q Z z Z z Zd
~ a Y Y
Y Y Y Y ~
tt~
U ~ p p p p~ Q
Q Y
D (~ Q p a p m n w ~ ~ ~ >
tm Q t~ z Q
.-.
N ~ ~ ~ ~ Q Q ---
~ r
w
0 o g g
N
Q
Q C9 C9 C9 C9 C9~ Z aoU
Z n
~ Y _ C L" ~C' CO w w ~ ~ u~
Y l~ 1~ Y
z L C C n w w
U '
' ~ JzN Q QQ QQ Q Qa O O .
n - p
~ Z Y Z Z Z Z Z ZD ~ a ~ ~ ~u
~ ~ ~ ~
tiA ,_ Q Z ~ ~ ~ ~ ~~ ~ ~
,_, U[ a ~ I~ C'~ J
J LL
_
J ~ ( Cn Cn V7 V) V7Q p Q Q
Q ~ N ~ '~ ~
A L"a L"~L" C-'~
~~
J Lv Cl.u Q Q U Y Y
Z- ~ g gQ g~ g~ g gU > >
~ a
O _ w ~ ~ ~ ~ 7Q ,m .= cntn
O N p z 'o U
O. N. U ~ J J J J J Z
O J Q Cl! Y a Y z z
a Q wz~ w w~ ~~ w ~~ Q Q a
v
oc C p~ U Sc U U U U UU N ~,, J Y Y
U ~ C ~ N
C ~~ "~ ~ 'QL'~ z
Q
O L ~~ ~~ ? Q Q
OC ';
~ ~ ~ ~ ~ ~~ n n D
a O O c ~ Y
n U U :~ U ~ Q
M
O
n
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0 CO o~0 ~ N ~ c0Q ~ ~ N
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C~ ~O c"~~ ~ ij ~
M
t O-M .
~ O
m r7 M ~ a0 N n~ J J O
L X X ~ XO ~
C' Y ~ 2 X
_gg_
CA 02290738 1999-11-19
WO 98153103 PCT/US98/10561
TABLE S (CONT)
~ ~
i
~ N Z a
. ~ N ~ N
~ ~ Ly
~ ~ ~ N Q
a d a ti N ~ ~ Q _ ~ ~ Z
Y N ~
d w ~ ~ "' ~ ~ a U "'_
w
"~ .. d r m c ~' O ~ " G~.. U
n U
d
'~~ ~ x cxn d U ~ c~u ~U d
Z
~ a .. a a d d
(~ d d m w z Z
N ~ N _ d ~ O G
Q
Z g Y ~ Cr ~ a a
z
Y U z = z ~ Z Y
L" N~ o Y Y z C9 O p O Dx U U
c
~Y w '" a -' z ~S~c '1 x a ~- Z Z n a
~ Q d
Qa z
. c _ d
,
U ~ ~ d Q C ,'- C
u
Q ..
u' n a ~ (.9 (~ U ~ 0 " U U
.. CL .. .x.~ a
,'~'
Z ~~ Z? VY a 0 ~ Z~ O O x
Z in Y ~ a Z H ~ d N N '~ z
(~ d ~O ~
Q Y c O ~v (' d d _
Z . n .
Q ~ V
Y Y N C ~ ~ Z
N W w ~ O x d d =
d j O ~ cr
O Oc OO Oz ~ ~ ",~_-~g OQ O ~ ~ U~ ,_-'
~ U
C ~ w O tn ~ Y ~ ~ w
U r- Y ~n r- d O Z
d a d d ~
n Y
a Q . x c U a
p 7 O O d d Z U~ d '~ w w d cn <n O
~u'a > C w d Q
c '-
. w ~ Z 7 Y U C ~ ,y p v C
u. ,.- ~ Y ~ v~ U w' Z ~ V U
iy ~- x d C = ~n w a
,... U
m .-
_
Z>Y ~Y ?d ~ d zx QU Z~ ma a Z Z z Z~"
=- Y U w w
v U U ~ cri m a ~c ~ ~ ~ r- O
C ~ ~ C ~ ,n d
'i Q N Y U J z ~ C ~-~- O r-
~ ' O N Y Lv O O (,~
~ O
g
~ Z ~- z w ~- O Q Q
Z Q O
c
Y U' w D ~ Z a 0 ~ U N
a a "' O a Q
C9 w O O
n. p x o~..
rr i
~ d ~ = X ~ ~~" O ' (9 cn ~ U (> U
d ~ C U w N
w
Ui = zQ O ~ '_
U '
c~ ~ ~ w n Z DO O D " y
.~ C a Y Y Y
O cL
Z N d ~ ~ Z ~ Z w w O 0
Z U C ~ d
oo
U N ~ ~ c~ O Q ~ iU Z ~
d ' N z _ g x ~ O
'Q ~ ~" 'Z ~=
- ~ U '~ ~, Oo. x ~ '.- C O
N U Z w Y Z N C U~ m cn
Z O uo C ch
0.Y _ ~G i w d~ Q~ Q YO O JdN v- Q Qa
E aY a Z ~ w~
G -,~, c N
C c n ~ N~ ~ x Z u' Z ~
nm 2
~ Y
~ Qd Qd QQ Y N ~ UU ,CyU ~ vt
- Z ~ .. c .
CJ x u
oc ~ x '~'a m z cn r
= Z Z ~ N x z d
Z ~
N p p O cn I
Y Y Y
ch ~,p O
~
C n ~O ~" Q ~ ~ ~ '~ ~ ny
p 'n ~ C
Q V ~ ~ ~ t~ ~ C ch ~ ~, II
~ N O
~
~-
~ ~
i >
m o ? ~ ~ ~ d t
p ~
u1 I
i J
J
CA 02290738 1999-11-19
WO 98/53103 PCT/US98/10561
TABLE S (CONT)
Z Z Q
~ yu
O Z
O ~ a
Z ~ U O v O
Z ~ ~ z g a
~ ~ d z ~ u~ ~ p C9
QQ ~ ~ Z ~ Oa Q O
W Q J m
YU ~N a p ~ Oa O
N
0 Q z 2 ca m
w w D Z N ~ ~ 'r-a ~ W O a
O r- ,.-
~ z=~ a C9
z U yu Z a w C
0
~ ,~, c~ ~ ..
Ur-Z w -,
c'~e ~ Q ~ p O d Q Y Y ~ ~ ~ O p Z
v Z O O ~ ~ ~ Y t~ ~ O tV
U
~ ZO ~ NAY O cv~ N~~cZnz~ N OpD w cv
U o ~ N ~ U a ~ a Q Q ~ Z '~ O N ,.~,, O
Z m ~ ~ p" ~ U ~ = O Z Z Z O a ui ~ ~ Q ~ a D O
~ " vg a =~ ac.or~o~ m a ~_~ z ~., °'m
O Qz ~ Zo z NZ pZZZ_Y~ ~v ~OVn. ~ O
t- Z~ O WQ u~ C~ Yr~-~.~-,_~..~can ua7n NNC Q OO Z
O - Oc O ~_a~O °OOO~Z ZN IZ~
_ F- F- ~- .- F- ~ ~ ~ O
U cma z a,z a °a ~aaaZ~ ww w~z U'
U O ~O U c~ gooo00 DO 00~ U ~~ NCB
Z Z ~ a N ~ ~ -p ~ ~ ~ r~ r~ U a a a a a. a Q Z a O Z
N ~ C Y Y Y Y Y Y Y Y Y Y Y
Z F-- U U .~'C F- .n U U U U U U U U U U U p w O ~ J Z
H OQ t~ Q CO OOOOOO OO OO~ Z c LO
z z S z g a C9
Lv m ~O Q CV w ~ = S Z Z Z Z Z N Z
oc m=oN Na s fn (/7NNlnNN InVJ tl7(n
O ~- ~- r- n- r- ~- ~- r-- ~- ~- r- O ~ W -
N CO ~= ~-v Y Q QQQQQQ QIG QQC~QU Z~ cp
~ Y d Q Q ~
Z ° U z s N v = U z z z Z z Z z z z =I= ~ ~ z p = r. U
P
OD a('u0 ~O M N Q
I O . ~ O' N CO rw
0
U c~ ~ ~ ~ ~ ~ I
a _
y ~~ J N p . n O~I~
C
p: rs ~ a0 ~ .O ch ~ ~ O cw N ~ a N ~' ao ap ~ N'
O I ~ n f~ n P ~ ch N ~ ~c~ N O ~, ~ Q °~ O'
Q~ ,OcV~ OON - ,OQ~ N V cOIP
C' ~~.0 D X ~ X f/7 X ~ J J X ~ X ~ ~ ~W fn O J O p10
-90-
CA 02290738 1999-11-19
WO 98153103 PCT/US98/10561
TABLE ~ (CONT)
C
O O ~ O ,L.
Q ,'~ 0 O
Z ~ can v U U
z.. O ~ O ~ U
I ~ ~3 Q O g z_
Q ~ ~ ~ a ~ p U ~ Z
~o a ~ N o
z ~ w
U ~ c .. ,$ ~ UO U c ~ w
nI.OO_ Qu' UU_
~O~ "-' ~"'O U~ .r
°° c ~ Z J J '.- U U U Z
pU Q ~~ ~O ~~ c ~ U O
O ~ J
~ w 0 °- a ~ U a ~ a
-, a .- <;, a r c ~ O J w
m ~ ~O ~ o O= ? Q o -_
d.
O Q O Q ~ Q a ~ U -~ a a U U
z ~_ ~ ~ U ~ G
a Q p N ~ O D. ~ r- G Z Z
"U U~~O z ~ a as
i '.'' '" z C Y ~G ~- z ~ m m m N ~n
~ d Z ~ ~ ~ tip O Q
J
oe ~ ~ U O I Q a >v n cUu Y '"" g a W ~u a
C i-- ~ ~u ~= p C v.. ~ 0 ~ x Z O z Q O Q m
~w Z v-u'-'LZ ~O'Q rC-Z ~ Z.=
C ~ L O yu Q Z ~ ~ m C~ Q O Q ~ Z Z ~ Z Z
~°-,U~yuz z pO~Q vp~ ~n"'.
,u ~ZCO~O '-d ~a0 O~~ O O-
Z ~ ~ ~ a ~ w O Z Q O Z Q m w" O U_ Z_ a Y a
Z .-
p_ O O = Z z Z ~ O Q J w ~ Z w tx C
Z °' U U O p p a Y a Z : O ~ z ~ ~ Z Z z ~ a ~ a a O
ce ~x C9 ~ ~ Z O 'l O o Q m N Z ~ Z U ~ m ~ ~ ~ ~ m ~ ~ (9
Z Z ~ Z ~ a "' -' o o ~ Q ~ Q ", p z ~, p m O a O Q O O U
m J O ~ O O c~~ '7d- V ~c Y a ~ n- g ~_- C U C m U U U z U U U
a Q Z Q m C N C >' Q
n U m U via a ,- U v ~ ~ a ~ c > ~ O m a Win- ~ ~ ~
N Q
i;,j . . Q °~ O
Wn I W O N Q N a
CDa~O' a~0 ~N~ Q
COO~~ ; C '~O'~
Y ..J,. ~ I J V ~ x . .
Q P N ~ N N .p
QQ ~~ ~~$R~~ R °~~P~ ~ ~~ ~ Q
~~~ lol J ~ ~ ~IQ r~ a~ ~ ,n °° ~ ~ .- ~ m O x I~ O Ix
' ~ J > > ~' 2 -f X ~ ~ X N
-9!-
CA 02290738 1999-11-19
WO 98/53103 PCT/US98/10561
TABLE S (CONT)
w
Z
Z m
U ' z ~ O c O
n.
v
n
w U~ c O ~ ~ = U ~ C
~ ~ " c a
n ~ U U , v .
C N ~n
w v C9 Q 'r ~ "
w
~ Z
m O ~ U '~?i m mi
'n
u- .: .. U = a G U
J co U ~. O U ~ m a Z
- ~ ~'~'-. Q C9 C~ C tt~v ~ ~ _
Q "
W i O ~.-_- C C O w ~ ~ C L
O
a w ~ U m z ~ ~ ~ c O
~
L
C9 O c ~
D ~
~u U. O .O i >- Z_ ~ c .= U eri C
,i
,- c ~ ,o~. = U ~ U riz c U O
U ~ v W
..
U QC9 ~ Z N'" c " ~;Q~ C U m a
Q = U
U r-~ ~ z ~ O c a v = Z ~O
~~ ~ m a
Q ?C cr ~ U ~ ~ ~ ~ 0
a
~ = ~
y y "v N ~ N J J Z LL
d ~ O ~-
.-r U~- OU .:U yz ~ ~ ~ n N = Q O
,,Vud. ~' ~ ,~ U u. m
~ Q
U Goo ~c~ ~-~ ~m rJ ~- ,~ ~_ ~ ~, ~ ~g
a G z== ~jZ Q UN U U _ ~ ~ - Q
j~ ~ .r C
~ ~ ~ , =U 0~ Q Q C ~U O ~ ~
Z z
can~ P vQ ,n,~n~~ chU ~ ~ U ~% umutin'~-'
~ ~ ~
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~'Q zZ ~ m '- w w N
~ _
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C = m~ ~ ~ O ~ ~ w Q
U
w ~~ ~ __ ~c=nC~ C~ '~ " J U
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w C Q
O z u ~
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ocm .u~ '.'.'ZZ QQ mm m<n ~ J ~ p . p Q U
'W ui O
,_ -Q ~ ~~ ZZ ZZ ZZ ~ rQ-Z D Q ~ ~ J J " Q
y Q j ~J JJ ~ O
~ ~Z Z JJ ~ _
Z G QQ JJ JJ JJ ~ ~ J X U cn cn O
~
X
V a.u' ~ ~n ~~ ~y-.aQ-~ ~ C Q O a cn- ~
'i'.. t~ U U z U O -
~ O
11J tLY N CC~ NfnCnN t/1r Q Q N ~ J Z O z Z-
v J~ ~ ~~ ~r Y J ~ ~ a
~ X O Q m
.. X g
.. ~ ~ o Q _ ,
~ a a- J UU U UU UU ~ ~ Q "'~ 'u ,~
Z U o
' = ~ U Z~ ~ ~ ~
' Op O~~~D~ a Q~ ~~ ~~ Q Q ~ ~ = wa
~ > > > ao
C ~ m
U UumuZ mm mm mm C9 C9 ~ OU a.~a w~ a....
C n ~ a
n ~a ~21=u QQ
I
M in n
I Iv U Q Q
Q
I~ ~ 'N C X
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G n' n O~O a0IwO~f7'~P N ' m c~~'7~
ch ~ a~0
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O~ X ~c~ >> >J ~J ~ J J
C9~ ~IX
-92-
CA 02290738 1999-11-19
WO 98/53103 PCT/US98/10561
TABLE 5 (CONT)
Q
D
O
J Z
Z z U Y w
Z _ Q V '_
. v~ z O Q ~ri
O
z ~ ~ Q~ U
'_Q7 m ~ p ~._ _~~
v~ '° u.' a- Z z p
O " ~ U "' ~' O
n, c _c ~ Q ~_ ~ ~ U cn
v ~ m '~ ~ ~ Z ii
Q
'' cCn . o N= O = Q D O " p O vri
f= m~ a ~ z ~ C, O O
Z Z V Q = g Z ~ ~ w w ~
W C ~ Z
Wn C m ~ r- U ~. a
U U ~ c~ m O ~ ~ a ~'.' ~ U ,Q 'n ~ ~ ~ ~
ZO m z ~ ~-Q z ~ Q .. O ~ m
a m n Ua ~CZ ~ z O~ O ~ ~ O a O O
O d Zz4 Q~p O ~ mZ QQ .. ~ cn O
O ",, ~ L" a cn C ~ ~ C = e~~
U ~ Z ~ ~ ~ o, "' u, ~U a U ~ n. a ~ O
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U O OJ = N ~y= O .. Z N " ~' X v~ C cn ~ Q ~ ~_ W umu cu m
U' Z Q U C ~ ~G a ~ u' Q m Q Z ' ~ Q v~ cn cn v~
C O m O Z d ~ cn Z r. p., ~-- c~ O ~ Q Q Q <f
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2 C (~ ~_ ~ Q ~ -~ Q C m d ~ ~ ~ ~ ~ ~ a ~ M W a u~ mn
,~ ~7 N U O Z w C uU-. z Z ~ ~ Q ~ U ~ V~ C N O Z ~
z "c_~~,~z U"'U O °N~~ ~a.o g o~ wmmz00 00
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a m m ~-- Qa. a wQw~NO ° -~ D O~~g00 00
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a L L ~ Z a Q '~ J U ~ J Z z Z '." O U O ~ N N N Q O O O O
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C~ i ~~ X N ~ X ~ ~ ~ ~ O J O Z Y -~ X ~ N OIL ~ .X~~
-93-
CA 02290738 1999-11-19
WO 98/53103 PCT/US98/10561
TABLE S (COLT)
~ ..
~ " '~ Q
,~ C
Lv J lL
O
O ~ g U = x ~ O
pQ ~ a ~ ~ J O 0. ~ I V J
°__ _ ~ ~ Q ~ ~_ ~~ O C~ ~C
°° Jo O ~, Jzm 0 '."
~n ~ c C J r" z p C ° ~ z
ow ~O az ° ~ ~~D
'-' Z ~ a Q
U ~ ° = U~ z ~ a Q ~ cn
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U DO U: V ~~- U~ ~ ~ ~ ~On -~'d uC.i~a
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U CV O ~ ~ ~ Z ~ U J Y Y ~ ~ ° O J y ~ X ~ ° C ~ Z
D ~- ~ ~ °- ~ ~- ~- u'' z Q ,y, C~ ~ d z ° "' ° Oa m
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o"'c ~ O Q ~ O m ? ~ D ~ ~ C9 ao O E D Q ~ ,~~~~ ~ O ~ D D O d 01Q Z
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C' c~ ~ X X X ~ OIT ~ X ~~~ ~iX~~ ° ~ °i~ X
-94-
CA 02290738 1999-11-19
WO 98153103 PCT/US98/1O5G1
TABLE S (CONT)
Q
C
~
> > ,~ can u,,H O ~ cn I
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-95-
CA 02290738 1999-11-19
WO 98/53103 PCT/US98/10561
TABLE 5 (CONT)
Z Z Z Z I
O ~ U
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~ oa
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l
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n
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Q ~ ~ .~. N QQ QQ Q V7 Q p~ ~ = p p~ IX~pImm ~p
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~
-96-
CA 02290738 1999-11-19
WO 98/53103 PCT/US98/10561
TABLE S (CONT)
X Z
O °' U 4
w ~ ~
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4 m d. a g a n
G_ "'~_S v z ~_ _ _ Qzl
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r- r ~ Y ~ CV
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Q Q
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Q Q a N U a N Z ~ C9 C9 p o = ,Z" w~
t9 0 = ~ ~ _ '-' Z 4 Q ~- a ~-- r- U_ z n. n.
m ~ J ~ m c C ~ ~ Q O O
ZZ '--o'~ = U ~,o"rcv = C9 ~ aC. a cOaa
cn O O Z p U Z 0 ~ Z Z ~ w ~ '_ ,-
OC U U ~ Q ~ m Q U U u.W > > > "'
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cc''a. ~ m N ~ G m ~ ~ U Z ~ a a = i i O = a cNn in
u' °° m U o '-' con '~ Z U_ p U C9 U' ~ ~n N ~ ~ a a a n N ~ c
o Z ao Z Z
C ~ U ~ --' U -~ ~ can ~_ "= Z Z Z a Z °° Z Z N C C ~ s_f
w r= ~ m r=z- z 4 Q j > ~ ~ f-~~ _ ~ ~ ~ ~ .r ~ ~
°~ ~~m c_Q4QC ~~m YY~~ 000~0~0 00"'"'UU
O OON,OU JoO ZYN ZZ44 ao.a.an.~a- a ~c~clNN
r
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O,Q 40,Yr, 00~~ n4.aa4,~~a~~a ~ V~~~I~,~
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m 4Q~ jpp~Z ~o~ zzzZazzIZSmZ =O44yuu~
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i
-97-
CA 02290738 1999-11-19
WO 98/53103 PCT/US98/10561
TABLE S (CONT)
~ U ~ ~
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-98-
CA 02290738 1999-11-19
WO 98/531(13 1'CT/US98/10561
TABLE 5 (Corer)
J
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p
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m ~~ ~ ao~g O O O 00 O
N-~O Lr C O O~ O~
N~ N ~Ow ~C
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O 00 = '- N ~JU O ~ p~ Op OD
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Z O O a u~ Z v Z O Z ~ N m m m ~ m Q- m
a a Q ~' N. a 0 N C QL" ? G C ~ N N N n N N N
U N w. ,_,~ Q ~- N ~- cW- r.~- Wu m Q m Z m p
Z ~mZZQ~u~~ ~ O~'~Q Q Q Q~ QC9 QU
a w (~ ~ ~ (~ N = G ~ Z ~ ~ ~ ~cj ,~,., c~u = w O ~ 4
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Q Z ~u m w >' w ~ uZ.r O z O ,,~,., r O ~C.. ~ () ~ O ~ U
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c9 c9 ~~zzz~d<n a. Q~Nz ~M ~ ~~ NU Op
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_(~(~_
CA 02290738 1999-11-19
WO 98/53103 PCT/US98/10561
~~AE3LE S (CONT)
J
W O U
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- 1 ~~-
CA 02290738 1999-11-19
WO 98/53103 PCT/US98/10561
TABLE S (CONT)
U ~ ~
W
O
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U a U ~ U ''- ,V ~ w w ~ O C p ~n
v7 ~ _ ~ _ ~ m 'v a N U ~ C .~
J Z J a J J J
Q ~ Q U Q Q Q ~ _
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' in ~ ~n (~ vi O V~ C9 cn (g Q C ~ ~"~ X
m
O" ~, OZ Ov a. ~~ '''OQNQ C9
V _.. ~ ~., O % U ~ V .. U p J ''' U p Z O
_ ~o a m ~- .. .. Q C
Q ~ ~ ~ Q ~ Q ~ Q ~ ~ "' ? J ~ N ,C Z J C
~C~ cC9 ~O cU ~C9 '.'.' c O m~ ~OmpU
v ~ ~ ~ Q C ~ X ~~ ~y Q C
~ ~ ~ ~ ~ Q ,.~ p ~ ui O = Z a
oc ~ ~- ~ cv ~ ~'? U ~ U ,o ~ n = Q 4 j ~ ~ U Q
V CO C(j CC9 ~C9 G~j ' ~ ~ m~ w0~QU
~u o. ~ a ~ a v a v a _ ~ ~ m ,~ z ~ O ,U m p
. <;' ~ c' ; 'r ~ ' ~Q ~ v ~ ~ Q w a u~ C z
N
tL ~ Z LL Q tll ~ LL ~ Z LLI Q CV ~
U in ~. r, N ~ (' ~ ~ . U N Q Y p O r- ~ Z
Q r- '~ Q ~ ~ F- ~ v Q C ~ X ~ U Q p
H ,~ j ~ ~ ~ ~ ~ ~ ~ Q ~ j w L '"~ ~ u~ Q N ~ N m uJ
4. v ~ _ _ v Q U fA ~ __ a IIJ
Q:-C9 Q'~' Q"~ Q~C~ ~:° c ''' QO Z,zu,~= Z
~ ~ '' ~ ~ '= ~ '- ~ '' ~ ~ ~- j U '~' ~ J U C9 m O "' C9
J J ~ -J ~ J
> > ~ ~C ~~ ~ a g0 =O OOZ~~vm O
O ~~ m O .. O ~ O ~ °' p .. v ~ ~-w.u ~ O p ~n p
u. a ~ p ~ r- C Y C >"' ~ p Y
N Z Q U'r Z m Z U Z ~ (9 Z ~ . ,.,, ~. ~ Q ~- z z = p Q z
.- '-'-' ~ = a m Q W
O ~ U v ~ O ~ U' ~ C9 v ~ C9 Z O ~ Z O N Lv O O Q m Q p
H U ~'~ U ~ U ~ U ~N U ~U ~ ~ U O Z O n. ~ ~~
J ~ 1- J ~ J ~ J ~ ~ J ~ O U ~ ~- _ ~ O O U ~ ~ pQ n ~.r~
C9 C9 (9 (9 C9 ~ C9 ~ C~ a O a ~. O ~ ~ ~ Z ~ a ~ ~ ~ Um u~ Z
Z d p a O p p p ~ ~ C J Q O O O Q U ch ,,, ~ O
~ v Ur ~ ~ ~ ~ N ~ ~ ~ U Q m .U U U U Z Q - ~ Q Q
i '
I
I,
O ' u7 N ~ c~ a~O r~ _ I (V a~rw0
mt0 P ~ Q~e~~NI~ app
C' VZ N ~ ~ ~ X ' X X ~ ~ rNV Y
-101-
CA 02290738 1999-11-19
WO 98/53103 PCT/US98/10561
TABLE J (CONT)
w
z z ~ ..
..
O
'- °- ~ ~ ~ w c o
Q (~ ~c ~ ~~ ~ Q
z ~O ~ c ~ a c
J n. w _ t11
~Z vZ vQ Z ~ d
~ C Q
U o, ZO Za r~ = a
Z ~ J ~
~
d d_ ~ ~_ u' a u' ~ ~ u' 4
~Q _~Q vQ "' Q m a.
w ~ aC Q
.. N r- n ~ ~ i ~ cn Z a O ,1, z
o_ Q ri m ri U ~-i U ~ z Q C Q U
U ~ ~U ,_. ,U ,._. a. ° ,~ Q c Q
XO v ,., Q .. Q O Q m a u~ Z
U ~ c ~ cv ~ ~! ~ G C C Z Q Q
p ._O~ ~ 0 Q? QZ Q
ch v c'~ ch ~ ~O uJ LL z J ~ V
U ~ U csi U i w w ~ >' O
m ~ ~y ~ ts~ N C c' U ~ ~ w
", ,.u a Q ~n N ~n ~.,~ cn p p ~ m ~
Z~ ~ ~,C c~n~ ~,_~,., ~ Q QZ pu=g Z
J ~. Q ~.. Q ~- ~ p Q
~ ,~i, ~ ~ r>--- ~ 2 m
Z Q
< i csi 0 ~ ~ cn v w .Q. ~ Q Q ~ O Q W
Z m Z
t.u x ~ _~- ~ cc~V ~ c i cu'n C
Q~ Q~ Q~ Q ~ ~c ZOr a- Oco,
Da ~ ~~ N"'Q' c~nQ ~ w w~ w Q~ O n.
,s, x ~ C Q = Q Z a = J ~ O 1L Q ~ w U u~ ~ cat
Ln O (W tL O O ~ C' V7 w p O m O Z
x0 x0 x0 c ~__ ~ O
H n. w ~ a a U' Q Z = Q n.
Z m C p O = O a O ~ ci~ m ", O
Q Z ~ ~ Q ~ Q J C -J LL ~ ~ = d J Y- ~ J (n Z Z
nQ.Q aQ.Q aQ ~ O ~w Yz~~wZ~
.n Q x N Q ~ o ~ ~ ~ 0 ~i a ~; p C p U ~ O p a ~ ~ U
m
O O x ~ ~ ~ y. ~ > z O ~ c Q c ~ p m z r -~ m
n C9 a nz. U~ n Q n Q n Q ~ ~ ~ a a d v s ~ v. z C9 ~ z ~ m c~~ c Z
~ i
.Y O OU O t~ 00 ~
m c o, ~ ~ ~ N ~ ~ ao ~ O H m
C I
m n ,~ '° c Q o ° ~1~>x~c~ xx~~
(' I x x
-i02-
CA 02290738 1999-11-19
WO 9A/53103 PCT/US98110561
Uncogene and Tumur Suppressor Gene Array
In the oncogene and tumor suppressor gene array according to the subject
invention,
all of the unique polynucleotide probe compositions correspond to genes that
are associated
with cellular proiiferative diseases, specifically neoplastic diseases. Genes
of interest that
may be represented on the array include: oncogenes and tumor suppressor genes.
In a
specific oncogene and tumor suppressor gene array of interest, the spots are
as provided in
Table 6.
-103-
CA 02290738 1999-11-19
WO 98/53103 PCT/US98/10561
TABLE 6
a~
a ~
' " ~ o
N _ ~ ~ w
Z
cd
O 0 = O L1l
U "'
m o g ~
I
E n.
I + r '
i ~n m u. ~ r O
(t)
y ~ ~ C
_ _
O M n- ~C I O
U .r . V
L O N _
,~ U
m O .~ U .~ ~a
-,
O is ~ ~ o
o ~ ~ ~
w a U c O c~ ~ v
,
a y Ut!~ ~ c CL ~ O c c
c ~ ~ a o > ~ m
.> - ~ z E w W a'uo E ~
, t
+ Q o ~ ca U c n
O = a N L ~ N W O
Z
U U Q o CL Q ~ o Y
~ ~ a ~ ' ~-a- ~. ~ ~ u.~ a m m
U ~ m L O O C O Z
C7 ~ Q m ~ w > LlJ
_
O ~ t~
0 U Z O O Z O a ~.r- ~ N u-. a~
~
~ = ~ m
o o a = a a O -a m m w O w ~ a. z
3 I- ~ m ~
~ u.~ Zti uJ 1- U - a
d ~ ~ Z z ~
U
v,Q ~ O p Z = a ~I a m U Q - o
F
'~~ >' ~ Z U ~ ~ Z -~ ~ ~ Z lw-~ ~ Z Z ~~C7~ U ~
Z
= a ~ t ~ o
~
w E~ z c~ a O O ~ zo m w ac ac a
O ~~~ 0 c~ ~~ u..CLCLU a~ O U ~-~ ~ f-~ a~~ a CL
' u
o ~ 0 0 n w~ m C9C~O rtC7 a ~ ~ O a f E m
a
- ~ - in U O ~ a D O ~ m z
"
m ICtU O U U m w ~ m ~
O M O w ~ ~U ~ Z Z ~ ~Z ~ D O I- C Q ~ ~ ~ ~ CL'W
~
O ' ~ ~ _ ~ . N U U
L ~
O m Y Q H p U U ~- ~ a H- J ~ Z y.~ 1L~ l L
m " li1J
E o ~ ~ ~ " z o 0 0 ~o ~ U g c ~ m ~ g E m z cc
~
~ ~~ " - U N
WO~z o ~ ~N ~ ~ ~ CL ~ uJo ~ a ~ a l W = w c ~
r ~ N m n
z -- w~ a ~ m z m m w m a a~ m z o m
i
U ~ ce ~ ~N ~'~~ a Q m ~a m i.i=cnrc om m ~ ~ N m a ~ m
= "' '~ w o m m ~ m
~ w im a a~ go m r m ~-u~m y c~w ~->r a > U ~ o uWm t-m
~ m W m -
t9~ 2iZ ~ ~ a l-I-f-H UH-U f-~ ~ u
,
N i
i
o
yk i ~ X _ t
X I
j
y ...r ~tO
C E I M ('~ N ~f~V ~ N C'~ C'~~ ~ N N COr
O ~ t'S~ ~ N (
C M C N O OfN N Otn ~ ~ ~ ~ ~ ~ ~ D r
O O O ~ M N 00O Or ,.~ N N ~ f0 1~I~ 00r M r
CO
('~N ~_~O COh ~ ~_~ c'7N N r ~ 1~-V
N O _
N
y j X X X X Y X ~ 1~~ X ~ X X I~ Y X X
X
( ;~ N ~
;
I
I I i
. I
-104-
CA 02290738 1999-11-19
WO 98/53103 PCT/US98/10561
TABLE 6 (CONT)
I
c ~ U Y I
~ O
O w o
N .cCC CL N h
N ~ ~ d
.N n U
W
tb .--. d m !z ~ N
a L o
o c H- 'm ~~ cn
..r ~ co tti a
o .o =~ ~ ~ ~ ~ Z
°' o
o d ~ a ~, Y
g' ~ ~ Zp L Z
w v ~ '~ a w ~. C, w ~_
N 111 ~ f--
N r. V V '~ C M O U Cn O tl-
' :° d ~ C'3 n CC d
U U p = ~ V V U ~ O U ~ w m
3 Z_ Y cu E Z ,~ a c CO m Z a
L L ~ ~ Q 'C .~ V ~ O Z ~ ~ (n z C
O vi = ~X ~ 0 ao Y ~ X ~ ~ 'a~ Z
o~ o> a ~ Q ~ > ~ ~ V Z o U Y ~_ o u!
m _~o V ~ OC c ~~ m Q ~ ~ y ~ _°~ N O
~°' ~°' p ~ a ~ .~ can ~ O ~? ~ ~ ~ _ ~ .o a
c~c a Zm ~a .~ YrZ z~Z~ mow can °~ Ew
Ca'3Q E= ~p c~nOY ~~jz~ Z~a U pUC~n~°U
v u1 ~ E a Z a ~ n- CG O ~ t11 ~ m U Z O X Z
iC U ~ ~ ~ ~ ~ Y O ~ O ~ p > ~ LZiI r ~~ Z Q f' LLJ d H
'-" ~ h- U z C~ _c. Z
4 Q ~ ~ O ~ cn Z Z ~- ~ Las. _r O Q Y U Z a O t- ~ O Z Z
Z
J J ~ O ~ O oC Z ~ ~ O O O p Q U ~ ~ p ~ ~ a o
Q a Z ~ ~ p 0 w ~ U d U Q a ij Z U Q O Z V Q Z n.
~ Q ~ ~ O ~y U ~ ~ Z ~ (O ~ Z u! = w O O N ~ ~ U w ~
Z U' t'3 ~ V ~ a IJ11 Q ~ ~ N F- O Z f~-. Z ~ cu a ~ r ~ D d Z ~ r CL O
c~~~L~u JY~ar~ ~C7»~»DQ~~Z H~cCaLZ~
(~ Z 2 O ~ Q U ~ Q ~ ~ ~ f- Z f- 4- V' f'- U ~ ~ N D a co !- D 14. Z U
~ M
N
N N ...
N J
C OD ~ ~ N vt fD t_~ ~~j h t0 t~ ~ h. N In O c_~ M tn O ~ tn tn 1
t0 O pp cp N c0 h O ~ N c~7 c~ CO CO N f~ ~ O N N C_O a0 ~ O O~ O~ 0~7
N N M r r Ch CD In M (O 1~ CO 1~ O ~ O N N 07
C N N n ~ O (p (D C'~ n r N M et (D QS O N In C~7 N C (D V O N
d r r N ll7 i~ O O ~ 1~ r r r N N N C'7 M C'~ M C'~ M In (O (D
C7 J J ~ ~ ~ X X ~ 0 J X J J J J J J J J
II
-105-
CA 02290738 1999-11-19
WO 98/53103 PCT/US98/10561
TABLE 6 (CONT)
C U C_
c U a~
... - U o
c ~ n
C .U Z V .C (O V
N a O - _O (00 cCC
N ~ ~ _tO
d N Q z Q d
f- LLl ~, N
U uj r
a ~ aC .r a r
E uy ~=. z
Z J L~_L O Z Y N E
Z U Z
p O 1- m ~ Z uJ Q Z
J
Q Z ~ _ Y Q U ~ ~'? ~ J U .= I-~- p
oC ~ ~ E ~ U ~ Q cn r
O C7 ~ N J O ~ O co ~O Q tn N ~
~- !- Z Y c a a
o z o ~ z _c"'n gz00 w °' c"'n lu ca ~N ~
UZ a~~inHrZ gY~N ~- ZZIZlI Z
-- D Q t11 Z C7 o Q
O 'd a ~ U Y r U U ~ cn O z O
Q J ~ a J W ~ r r f- ~ ~ LLl Z LLl O O
OC Z Z d ~ 'o O O a a p = C O ~ O Z n O w U
m a s
_ ~ r U Z ~ Z 0.. J ~ I- Q ~ ~ Z
O U
z ~ a z uy cpn z z a c~_n ~n m v~ a ~ a w
r v~ cn cn V O
O Q w z arc ~ ~ Z~Z~ Z Z U m ~ ~ ~ w z p a
F- I- lL lit ~ Y Z Y Q LIJ LlJ = Z ~- ~ ~ I-- - Z
O f"' O lu f- a ~ p
ct U ~ O f- " lu U O O Z C7 u7 Z_ til ~ O cn
U p a ~ O Z w p z U ~ O Z J Z z n. ~ Z
Cn Z a UJ ~ IL O ~ Q_ Z Z d tlJ U ltl U 111 O Ur Q
Q ~ z z V Q O a~ lzu a Z ~ Z O O a O O U O O Z ~ C
CL Lll L ~ ~ (~ m ~ n Y t- 111 ~ ~ ~ Z O U V U
(] O O Z Z Z C lit ~ O a C O ~ _ ~r~ ~. Z r N Z O ~ a O
p O Z ~ O O y p Q U rL o O ~ ~ O ~ O O O O O p O Q cn
~ O
Q ~N O
d ~ Z X Z ? ~ ~E -~ O u. Z .~ O ~ ~ ~ ~~ = a ~ ~ z ~ ' lj 'Z
m C~=aEUa~~'ECYncYn~-°Ua~la~.UU~c~nU ~~U
CS ~
v r
M O N
N cVD OO7 O
x ~ ~ Y
YI
CIO~OIOctNCD(O m C' C_7lrOMntn~cD000 .-cDN ON~N
~ 1 r II In I WY ("7 CO !n C~ t~ N M M O In OD r m C~ O a7 C CD V OD C'~
W 1 cp N O O N tf7 O N N CD O N CD '~T V 1~ CO N Q7 O p M
C N M N C7 I~ 01 f~ N ~ r N ~ ('~f CO Q7 M N M ~ ~ U'7 ~p cD
G1 tD 00 OD 00 O O O O O r In O O N O ~ 1- O 1~ N ~ N f~
X X X X ~ ~ Cn X X IX O ~= X X J X J .X
ii ~ ,~ " f ~~ ;
v I ~ ,
i I i
m
-106-
CA 02290738 1999-11-19
WO 98/53103 PCT/US98/1O5G1
TABLE 6 (CONT)
U
O
m
a
O
ca a ~ -
a ~ .- a
N a
N ~ w U C
. z p w
.~ c >- ~ a~ w m c
Z n
p _ cw cwn a'f.. ~ U a-
p .~
U ~,n a a O c d
U f
z .c m ~ - c
iop a U
a Z Y Y Z d ?- O
Z Z H ~ a
(n I-
W u w O a~~ _~ U a o
m ZQ O O a " m m ~ m '
.- wa = w t- z
C -
a
z~ ~'p a a ~ z z w Z p ~ a w
w w LtJ ~ ~ ~ U .- .-. Z U Z cucn" w
w aQ z z c~ cnw w ~ a '~~ ? w r
J J J
~ Q
a ZO O O ~ O Q Z N Z ~~ ~ O
Q
Z ~ '' ~
Y ~(n~ ~ w G. Y w ~c Y E... UJY Y Y
D.
J Y ~-~- O c0cC U (n I-J J J
w w (O O z Z y m v'o ~ Z O OU U U
.
i-
w
O C7w c C~ ? O Z V Z d~ N Q ~ z ~ pQ Q Q
7
~~ a
aH ~ Q ~ U a Z ~ N~ '~a a ~ w ~ HY Y Y m
d w O Z o~Z ~-~ w a acoo ~ w a f- a a o
w m O ~--
E,pJ a O N w ~ z a ~ "r " ~ z z a z ~z z z s
o
Z O O z O cnZ L cnZ cncnY U ~ m~
a' .,.
~ cn
O
JJ UZ h--~ f- 'J Z O -~a Z N.-z Z O i.-.a J O O Y
d U ).f=o oa~U w ~ U w a aa o~a cccn~ 0 (p~O
c ~ Z U O I-tL~ ~ p
a~~~ w CL~ ~ >-p 7-~ IL ~ fLCLN CL~ a w a m !-wn.a n.U
~ ~ w m
C7
c~' Z2 d ~ a U a t-U m E--u.u-w t-E-
U u.
.r
I ~ i ~ i
v
0
N
I
C 1a0O O O N N a0~ c~CO CCf~ N M 00 O ~ ~ VfDO N
' ~ O O
NO O N N O O ~ ~ tnOO N ODM P M O r'M CDN N N
O f~GD I ~ N N
O
N IN'~071~t~.QO O ~ M I~ 1~1~1~~_CO01c0f~ O ~ O f0O Q7M
M~ tC1Q70 M C O ~ N cDcD CD ~
~ M
_ f~ f~ Q'0 O ~ ~ t1700CD N ''M N N N
~ ~ p I ~
~ g g ~ ~ ~ ~ ~ j~X XX X X X X IX N JJ J J ,
I i
i I~ rI ~ I
i i I.
CA 02290738 1999-11-19
WO 98/53103 PCT/US98/10561
TABLE 6 (COt~JT)
O
m ~ ~ w
o a
Q N
Z
m
Y
Z tn U Z
p _ o
N
w o
V a
t '~ w
w Z_ ~rjop M
R Y
Z w " g m Z e'>
U Q ~e'r _ U -~' U
~ w
'
U N H c lLr w
v a
Z_ o ~ N ~ M
U J ~ c ~ ~ m Q
(O
'~E c cnc0
~ Q
"
o x Y Lr a N a
Q
' z
- v = a n
.
~v~Z ~ Z c T;Z fn
r U ~
Z
E ut z O N a~ n.Y O
Z ~ ~.c Q Z
F-
.
Y _ d tlL = v o Z
cn
a J ~ ~ c w ~
E ~
cn O ac U
U c Q Q c a ~ U zN .., ~ ~!-~ ct
~
V n Z , ~ Z ~ ~ IQ- ~ o a. O D
Q ~
Z ~ ~ I-" w ~ Z
w V Y Y I- ~ Z Z ~ m Z._z Q Z
~ j Q >
w E c
O O w tn U
Z O ~ ~ ~ cOZ m Q~ U Q
~
U z Y ~ Q p a Z O
d
w O c9 U a Z = w ~ ~m w -
N ~ n.
U g ~ Z Q = p n-ni ~ ~ Z Z ~
o ?
cc ~ c c
w Z w Z p ~ ' InQ O IL ~ O ~ Q ~
Y v (n ,
Z z O a r _ J w s ~ w m z O ~''z~ ,c~ ~,~ U ~ L
a ~ ~ ~ ~ Z I
. '..-X = C~ Q Z ~ O LOJ ~ r 0 ~~ ~ InS ~
w U = ~ Q O U aO ~i~ EN m ~~~ ~~ riE
~' U
'~ ~
C U r O Q U E E = ~ = ~ ~ ~ N
n d ~ I
C~U m laQ ~ ZU ~ ~ ~ 4 D ~ U ~ ~~ Nw ~ ~i'.E~.?,Z '~~c u
a~.i . - t J
-
I ~ v
m v
I ~ m
i
a I U
C C tn M NM MO CD N IrM ~O NI~
CO (DO r ll7N(DODCO O N M ~ N
M M OCO M
~ (p. ~ I COr O In O (OM_M t~ r COM 001f1!~0000CD O''O~tn~ ~ M
M i CDM N00~ '~ G0N O Nr O ~
I~ I~
~
M ~~ OM V( f
C N 'QCDM InO~ M h O r M ~ r m n~ NN N CVM NN Mf~01O ~
I M M ~ ~'~ O M
I ~
41N N N I=~ __ ~ ~ _ ~ _ ~ ~ = XX NJ J JJ JI~~I~~I~
~~
V
I I
CA 02290738 1999-11-19
WO 98/53103 PCT/US98/10561
TABLE 6 (CONT)
I I
i
I
~ 1
~,
U
I
c?
i O
O
I ~
z
a~
~o
U
~
O
I
Z
w
Nf~r CL
a ~~ ~ ~ a ~a
O
w H~ H w E
U V O
na n ' ' z.
. . aca ~ ~
E- ww w a
Q Q U a
4 X ~ Z m
ZZ Z Z
Q
Y
a
p YY Y p ~~ t
p il
M ZZ Z ~ _ ~O 'v g
w ~ ~p ~ CL
d ww u~ ~~ w
a H f- ~ cc~'O ~ p
~n
_ c'~Ot-O Zo O LZ ~ m
d O ~, tn
U
oN m aa a ~ ~ o0 r> ~ ~
~
O~
t pa =
_ c titw lit Zc c w p _ a = w
n
Y ZZ Z yC d~ =1-~ '; _ ~ Z= ~ lZ
+IZ "~c p ~ ~
~
Z~ Z Z ~ Ot2~a U-.- U3
0 o ' U ~ U z w 0
l w o~ a ~ ~ ~ ~ 0
sD OC~ __ = c . ~z ~~ E~ ' c ~ O f= o~IZ
a ~ Q
N ~ ~ ' LO CO C OC . O m O = Z ~ O
A I Y ~ N'~L - E'
~I7~a OC ~~ ~M .G.C ~ ~ 'XC E00~ ~ ~'N aa L fn
I~ ~ '- co p N 'D
~
41Q7>>i N ZZ Z ~T O ~~ UQ ~U nO OU ~ ~LL~1~~ ~ Ow C
O
N
C -NON, E--U l t'j
L- Z m m~ ~ ~p IE>-~a~.'E O r~Z ZN ~,w U U ~ Y
iLlm ~'
d N UIN~Y ~v>ww wCC~~ ~n ~ UZ m t~nvZ S ~w .nlnZ _. ~
CJ~~~~IvIL tcOCncO
I
c I
~ I
I
~
a _i N Q7~ 1'M ~DOQ~e~tnM (_OO I~M (D(Of~O O
O ~ N M N~ ~l'I IC7
~
~
A c~CD(O ~N ~C CDO (~N ~ OO CDt'~7~C ~~ GDN VO O~ ~ ~n t tn
m ef~ ~00 c'~D Mtntnc O OO O~O~c0D Nv Otn.-.-1~O ~ 0~ ~.
NO C~ D CD OItM '
I Q
C M c0O f~p fDcD<DV aD_ m IILLaON tDI~00aDM0) 010 _
N C Q~ !OC CDCOtDh XI~a aa 10p p> >> >_ ~'X X~ ~ _= Y ~
CJp =!vM IXJ XX ~XX N IX L
Ip
J
I, II y
I
-l09-
CA 02290738 1999-11-19
WO 98/53103 I'CT/US98/10561
TABLE 6 (CONT)
E
O
H J
J
m
Q
Q m ~ ~ m h
-- ~ z
Q m U
z W ~ ~ H ~ m
J ~ U Q n. Q
Y UJ d
Q aa~m 'z N ~- o
_ ~ Z 'rm o Y
N O (n I-Y
d. p ;~-- ~ Z a- tn ~ ~ p
n. d O O a ~ m Q o
Q o V ~ j ~ ,~ V Q t a cc
c%~ cn n. m Z i ~ cn ~ o Q a
z cn .r
Q m ~ Q I- .-. Y c_~ ~ Q .
U Z Q ~ ~ Z ~ z ~ ~ p o o E ~ Q
N
c U O a ~ O N ~ n. p N c V ~ ~ Y
~ 'd ~ ~ p~ V~ U O ct = can EQCZnn.
;e p, o d o Q U ~ f- ~ Q N V g ~ c~ ~ m Q
N > s Z_
~d Z c ~ 4- I- 1i ~ ~ m ~- Q fn d LLl > .c ~ ~
c N ~ Q~mQ_I U IL Q aO o -~-Z Z
_~ ~ p ~ Z x ~ p p Y p, ~ ~ 3 ~ I~
o. Q ~ .r Q Z ~ Q j ~ Q Z Z Z c d ai _c n. O O
Q c Y Z Q ZmC~ Z_QQ~a~_ ~v~~
io ~ ~, a~ ._ Y ~ V Y O Q Y ~ ~ ~-- C Q t1J ~ Q U. N.
O o v w Q J yu :_u O ~ a U Z °' a O w
t-- ~y Z Z Z ~ ~ h _. ~ w Z
c m °. m iu ~ ~' u~ Q f-Y- o u~ > > a E cr a oo~ o~ z
Y Q :n w ' ~ o Z ~ m ~. 1- p p o ~ ~ . p Z
~ p O O yn O tn cn pn U o o = Z_ O
a Q a O. ~ QY., o ~ Q Q ~ o Q ~ ~~ Q m
m m w " ~ OC Lu Q m ~ w ~ ~ ~ .c ~ ~.,~ s o
N Cf ~ O. ~ fnllJQZ~fnCn ~ Q1Q N N ~
d-- '~ ~ ~- a~ O~ZQOQQL ~ Z.Z U N. Q Z U f'- _
C ~ ~ ' vi N ft O U ~ tL Z Z ~ U C~ p m Z ~ ~ ul ~ ~ Z u1
01 Y m .D N . ~ ~ >' -
Q m Q u, .~ H- N. tn ~ !- Y Y '0 1 f- N Cn U CL p p a I ~ I c ~n p ~ cn
I~ M
o ( 1 0 ~ co
0 0
_ ~ ~ m
M ~ ~ ;O J
(~ ~ .~. x O
Qf ~ u7 CO a0 O~ t' a0
I~O~(~~7r(MDt~I7~ M 0~0 (NOONmNMVNV~m ~ ~f7
m ~ In p ~ O O ;~ N cD f~ ~ ~ O cD ~ ~ O t~ C~
C f~ ~ M lp V 00 ~ ~ ~ O 1 O ~ 00 t' O CO C Q7 C'~ N O V (D
a1 CO ~ N O N CO p M ~ m t0 O N V V lO (O
~ fl) Ip ~ p p J I~ W Q ~ Ip = ~ U7 Ip ~p W I~ Ip p IX
I
I I I 1 I I I ~~ ~ i I
I ~ ~ ! I
-1 l0-
CA 02290738 1999-11-19
WO 98/53103 PCT/US98110561
C'el!-Cell Interaction Array
In the cell-celE interaction array according to the subject invention, all of
the unique
polynucleotide probe compositions correspond to genes that are associated with
cell-cell
interaction, e.g. cell-cell signaling. In a specific cell-cell interaction
array of interest, the
spots are as provided in Table 7.
CA 02290738 1999-11-19
WO 98/53103 PCT/US98/10561
TAaLE 7
I
I o
I
O . N
cp0
Z_
~ '
O m _ cn m
~ e-Y
U 'oa ~ L
U
o ~ Z ...~ a Z is I a
-- c I
a ~,y E ~ E z_
I ~
O
U ~ >Z. ~ C o ~ Q ~ Y
~
ti .- C7 _ co U - a
~
z p ~ m ~ Z c ~ ~ Z
m
U O m m - ~ '= a~
C7
a o ~~ O ~i r ~ a S m
Y
. a ~ a ~l Q
'
o ~~ _ v U o t o o y
tL v ts ~ ~''~n v o Z
Z U a
' ~ ~ ~ ~ O ~ . Z a "'
0 ~ r fL
~ > N t11 Zrr' ~ LL o co
!- O
cn t c U E f- _D ~
Q > ~ o U
- = ~._ ' N
U ts
U
n m ~ ~ a x_ .
v ~ n.c ~~ . ~c . D ,- ~ U
N .~LL'tnWa ~ ~ CC L co Z
U7 ~ LL
O
C to O O._~o ~ ,,;~ --
a c
O E~ c ~ ~D ~ Q' ~.>o, U ~ N to
U n.
ati~U ~ ~a a ~ u.o a.~ a~ O ~ ' c~
O m
UZ ~ U '-
d ~tu= tUuQ tWO w ~0 ~.?c c cn O D~ O
t
E w 1- U a~v a ~ o a
E a 2U ~~ ccz ~ ~
a
Q J p ~ o z z ~
Z L
~CL~~ Q ~(OUJZ~ ~ a fL= C 3 ~ O .C Z Q~-
O ~ ~ ~ r ' ' w
~
~ aa aa ~ ,; E -o m ~ oz =
~ Z Zn- U ~ c~
Z
a>'UU O ZY ~~ F-N ~ to m-~V = ~ '~ O can
C9 Y ~ Y t n = D
aQ Q>-a CZ a Z Z i 00 ~~
I
tiLL=J ~ ZZ Z~ ~ ~ ~ J U Q yaZ ~~ ~
o ~~ l ~ ~ ~ ~ zx acaC Z
m
~~ U tut,utup zc~ Z r ~O O z ~ .~Q ~z a
O o
~ OO U I~OO O~ O ~ j0 O a ~ c z
~ _ ~
a
a c=c~z~ z ccc~a~a~ ~ w o I z; a~ w w ~ ~ zz o u.~
UU u w o ~ ~U wU U Q a ~ QcUn
u I m
~ ~
w tt O~ U ti~w ulc _~
m n
I IO, IzZ_Z_Z_z ~ z ~ N~ ~~ fL U ~ U~~IZ ~I~~
~ a ~ a~ O U
~
Z QCC=J Cnfn(n- ~J J J a C (~ t _F-J
I IO Y O O o V Z ~ YJ = y.
~ o
O
~ OO ~- O~O O_ _ oI~ p m~ J oW a m ~ U
~ I~ O~I- Wit'-VN C'~ N l(/)c'~Iet'N. ~ I-B ~C7~
~ ~
J ~I~I ' ~,~CC~ I U ty~ '~U ~~ Q _ z JUJ W- Z
~~ ~ ~ ~~ = ~ I E c '~ U
Q
U FIiiI-> Hl I1- I ~ UIU U ttf I
-- - - - .
I I I ~ M
~
G ( N ~ 'n I
O - I
N N
1~
aei ~ O I
I
Y I~ I r
C I' t~ O~ r'Qf ~Ttn V' COOCO~I I tD M N C'7 It~~f7
R tn~,~ ~ in O ~ I TINCQ N tf7~ 1 h tCC~GD
GD fD tCIIOO fDpO t0 IIn~I~ ImO
I O MIf~
M
m r-~~tO OICt N~~."OC~I~ N OO ~IQI n ~c'O.~~c'~~~ ~~ ~~ ~-~tl
lf')tDItOIC011~
C C9r~N mItD Itn~ C'7GOCD M N I~'IO~ h O M f~I~ ~rN IC~ICtD
N NI~t OaD O 1v00 O) O ItDao fOM
y t~~p~.- ~r- f~f~'-Otn M O OIO~ O MI ~p ~ N OO Ir
C7~~!X~p I~~~ IXX ~~~ I.~~' JiJ J ~~ I~~I~IX D J '~I7 1J JI_J,J
X u ~ ~ X
I ~ i
I I i I ~I
~
CA 02290738 1999-11-19
WO 98/53103 1'CT/US98/10561
TABLE ~ (CONT)
c .r .a~
o ° o
O N Y
Q ~ c z v
cV ~ w N J C
1- M ~ ~ O O in
U M °' o ! H .~n
cj N ao O o ~ U
-o v co Z U a~ ~ ~X O
U N O d E _ v ~ Z -'o
0 0 0
°' O ~ '~' ° co N E U ~- E
U ~ i Q
cC0 ~ C= ~ C '~ ~ w O ~ d L
CO Q O J d .~ ~ U~ d
o w ~ E" -c d U U c4 .U f_ O w C
U °~ ~ Z ca y ~ °' - 'o O ~ a ~ °'
a
co O w g '~ ° ran c I U '~ 0. QI ~ o d
m
D Q a ~ c ' N Z c J U[ U p o
U 7 ~ N ~Z~_ L O~ ~Q w o O Z
Z F- ~ ~ w C7 U Z ~ I- p ~ ~ O w Iw-
w U d O ol-~u~ a~ Zw ~w °~~ ~ a O
(~ Q > f_- d Z
m ~._, a~ Z E... m ~ Q ~ ~ ~ Z U w n. ~ E- Z Q d
E Q ~C7 d z~lZllUwnm. ~ ~w ,~v U O U O OU
p Ezmwo~ rtm~~op ~z as aw ~'~~= Q z
d OC7O O=ZOCm_ m- ~-O cI wZ o.5-0o Z w C7
d Q o l~ 1- O !- I- Z d ~ a ~ d w w ~ °' a ~ ~ c~~o J Y O Z
c9 > ~ g ~ z > > w w cn 0 = L = m a ._ ~ m ~, Z J
~=sn.,....~E... E--cn~p-o,°oo ww ~ >-Ig 'ou~w
U~ Icn dUw~d d ~_~~ ~wwUl-U ~ m
V w d r Z ~ Z Z Y ~ o w ~j z ~° Z au a-iN Q ~ Q ~
wIZ ° 0.. ~ O ~ N ~
Q ~ "~www w~n.OQUZ~, ~~ of a-d Zw°U
~ Z U c w C7 d C7 (~ ~ ~ J u. ~ Z w ~ Ei ~ICC zl_ Y w ~n
m O ~ ~ O >' O O >- w m E- U Z a, ~~ E- O U J Z ~ ~ Z ~a~ ~ ~ w
r-~= c_zz~Z z ~''°gUCC and °~ ~Q ~ ~~n cn
Z a~_ w Zm~cnmww v,~ m ~a~0 0
J~~~Yfn~Cn (n0-a_~M,JU~jS Nv oIQN~(AdZ''JQLt.~~ NQ
J a' ~ '~ ~ g ~ ~ ~ t~iJ o O~LJ11~Q N 4 D D ~'IU °Id >- O w U O m y.
o CL U
U~l~ 1- o o n. F- n. a. cn U U U > w U UIUI~.-~ U;cnl2~UioiZ ~ ~ F-I~ H-~o o-
co I I I i ~ ch
~ n
~ N I t~0
,r ~ _ x, ~n I
Y ~ I r,
c ~p 0p N nnINnOet INOaDih-nIOCDQ110r- fDOnON
~1 ~ n ~ O O O O ~~000 NII~D O ~ IQ1 Qir-icD nI~DItnIW nW N n r-n00 C~
Nn~~T ~c'~ O00 M I070cOC~lnr-IO1a010lc0 MG~c'~Or'N
C O tl~ Q ~ ~~.. n ~ N N NINIt0~71f'07 ~ ~ I~ N tMOI~ NIOIOIOirI~ ~ ~ O N ni0
~I~I~ ~ ~ ~ ~l~ ~~~I~~~ ~ x ~~I~I~~~ ~I~~~i~'»x ~~X ~ J~X
~--., W i
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I ' I
I ,
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I ~I
-113-
CA 02290738 1999-11-19
WO 98/53103 YCT/US98/10561
TABLE 7 (CONT)
m
g~
z
s Z .m U 2
c a ~ ~ = U
m E- -
d ~ ~ a i
a, a c? a a
a .c O m -i
o m U ~ m °~ _ a cu
V ~ -~ ~ Z '~ Q ~ ua.
:. ~ C~ - a H ~.
.~ La Z ~ F~- Z m Z ~a
O
o ~ _a ~ ~ Z_
>_ _ '° p I O ac O Q
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n _°~ a a ~ O f- O
uJ v O U Z
o a U W ~ >
a~ Q O 'c U
O ~ Z _
co m O ~ ~- O '~Cc
d N J ~ ~ N N z (n C
v IC _! 111 ~ ~ ~ 111 O
y C W N ~ _ .C m = in
3 U ~ ~ Q ~ ~ s
L O ~. ~. L1J N Q
H .O Y Z Z N J ~ o Q LU ct1
~ s ~ ~ ~ ~ ~ U ~ m ~ U
n cc
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z ~ m n. j c O
m ~ a
c9 z ~a ~ m _~ ~ .a a
Z ~ ri ~ ui ~~ m X ,°n p V J a°o Q c'~a
O Q o = °' ~ Q Q ~- .n u! Q r O
1- -Z n ~ ,~r~ '>_ = 2 .~ ~ _ ~ :Y N
a U u~ Z co m Z ~ C
~ c'~u t s,L ~ ~ s a can .~ m ~ Q s ~' s c~a s s Q a~~ 'n (~o
Z CC ~ ~ ~ m m ~o U Q m z ~ m ~ .n Z ~ ~u ~ ~a ~ m m ~ ~U I- a. '~
-~O ° ~ c C c c O r> .S I~ c~ .S S S ' U ~ .~ O .c c c ~ O- a IQ
. .- - '. Y ~ ~- ~ LLJ
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.
V Z ~ ~.cl.c c c ~ ,:~ c Z U _c c c c Z ~I.~ u. .~ I.c c c Z '~~ Q Vin., ti
I
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t0 01 I <D ~ I~ O N l17 (D N O ~ N p r
A lf7 M I N pMp r r O O M O CD r O O i f1
MAO p~tnM~I~ .Np~ I~QICOO~fOD ~I~cOljiaD~I~tC70f ~QIi~~N
r tOO~pGD~N ~pt~~' lnOlNMr NAM OC~~ NIO~DV'O ODI~' ~M e-
O OINXI~OT NXM MW cD1~00
'" M ~~ XIX X'X JXIX >- DID ~ ~ ~ X
C9 '7 ~ I J ~., ~ J W
i i
II i i
-1l4-
CA 02290738 1999-11-19
WO 98/53103 1'CT/US98110561
TABLE 7 (CON-f)
O
'oH v
c ~' N
c CO Z
o
_ _
Z ~ a
N
o U Z
Q Z Q Q
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z w
'
z'_ w Z
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n -
~
. o a Z
U
c ~ ~ p a
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U ~
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~
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c
o ~ Y r w L CLp
m ~ U 'n
f1
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O
U p U
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m
m
c o Z i ~ o U '~
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~ 'd-- Z
Z Z o O ~ A 4 0
w U d
~
C ~ ~ ~ E' y y (~ a ~ Z
a
~
S ~ rnE'~'>~ Z u, a- ~ c O Q
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Q i O a O a~ U c U
Z E-Q .~I~Um Y ~ p a p ~~ ~ o.c~ a m No cj~
a a
wO c m Z O~ t-C " ~d ~ ~~ ~ ~ ~, O m
m;_a c O o a ~ .'m
Jw ~ ~
U Z Z~JZ~ ~G(np~ ~~ Z~ d~ ~ sf C C ~p _.-N
~
a z XizI~~ -ow c~a 'w Z.c~ o- i
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~ ~ Oy _j~0~c~ .cp o Oa~ ~ E ~ Z ~ZaCm p ~~ ~l
Z U OIZz~'Q. ~a a.D~ ~ w ~W ~ _p ~w w
= co
~ aa ~ c J =~ ac?_O ~ c?a~a o ~op cv a ci.c~~'o ~
~ wl~~'ca ~O c c a,e n. m
o o
. Q ~:cnz~Q a~~ E~ o~ Xo ~~ ~~ ~ a ~aQ ~ ~ ~o Ea a
J E- J '
~
tuw >-a Z: c 0 ~'= a cad U ~UU ~ g gL ~nU U
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a
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c w~ ioa~N m ocWOn car~~cm c~r~NC.~~d'rN o 0 oao..en~
e- COP~ r n tnI I~ In
O CDtn
' O~p r~~tll~ON ~ COON ~O00~fCn~7~ff00 N ~ N O ~(7t0~0,00
CD I O O T OI (.~47 tf7O fDI ~
N ' t(7 O~O tn0 c'f Q
~I~ ~yCOO C Or rOO ~ 00
N CO~Wtl h~'~OOat~ tDC0r1~00r ODO l'~N7NX ~"~tl7CD
' at~ ~ D O O~O
CJX '~'p lt~ > > >> JX 7CJ X~-NN
~
~
I I
-I 15-
CA 02290738 1999-11-19
WO 98/53103 PCT/US98/10561
TABLE ~ (CONT)
I
ct z
w
~ m w
z
a ~
a
U Y ~ U
Q
a Q U tL
Z U ~ ~ ~ >
ac a = a. z o
z Z cn
o n z
O N N w O y u
cn
LL a = LI 0 ~ 4. a ~ O
J
~ ~
O
~ cn
Z J U ul ~ a
~ U
O a UJN J
~ a a
~
C ~ Q a ~ U U
a ~ ~ ~ N ~ J
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U ~ ~ n
w ~p . Q ~ ~
~ m z_a ~ u~,aac ~ z w ~ p
r1
utZ m w p N ~ H w
Z Y w
~ U =_H Y U c a~ O
~ n
Z ~ Z ~ Z ~ j w. ~ p
4- a''
m Z D g p I~-~ ~ u! w c Uo w ~ Z
Z z w
U a ~ m O O ~ N ~ aL ~ ~ CC
-~ m C~
~ u.r o F-as c a m T ~~ o f-~ ~", gy m m
Z p z ~ ~ Z c f"'O Z YL N ~p N U U'
z uJ . ~ Q
n Z Z o a a Z ~m ~~ gm ~ Q ~
Q ~ ~
Z Z p Z ~ 4 sX ~o a. ~ > U ~ N
Z U o p
p c ~aocD~ ~~ ~> ~ ~ ~ ~~ a ~N oU ~U '~c c c'- 1-
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urzQ z !zp a =, ~ O ~ O ~ =z z~ vzlz~ ~y ~,a~z y cl
,~ i' Z
N
U J . dZ m .-~~ ~ V (L NO O O~ OI
m w m Q ~ 'L w E ~~ Z~
~
~
Z u ~t~ z ~ a~ ~ ~ ~ o mo cn Q
t u j _ O N = ~ai=c?=~o~ ~= ~cd ~o
~
J O p ip ~~u~cn~ ~ o~ ~U C7O~ Z'~ ao 0 0~ .. ~
w g a w~ ~ Ng ~ v_ a~ ~ ao ~y 1- Jt c sa E~I~'n~
-' az
m a a aa a U) Nm Øo z _o vD L UL ~IH-!U a~ . CCU ~~ ~n~0
U U U IU a ~ -~ H v a CC
= a
I i
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I
N
- plp O r'N MO CDOftD l~f~N eth f~pO aDIlt7ir~ ~~N ~DMCf07N
~CD , ~ ~ ~ ~N r-N GDNr-NO aDNtf70tOiC0 ,ODtf7NO 0a0.
tt1N00u7 ~ ~ ~" 0
m OOIN1D ~~~ '~ r'OIM~ O~ f~O~ft0aDU71tDtnMUf;Of ~N ~ O)O O~ 0
~OiO M ~ p T _~ ~ 0
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N I ) ~f '7Lf D I ~ O Q ~ .
~
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C3J IJ B ~ ~ 7> >X X X JX XX X XX IX7C;X ~X X >-N NN a
> J a
>
I
-1 16-
CA 02290738 1999-11-19
WO 98/53103 PCT/US98/10561
TAE3LE 7 (COTVT)
Z
w
H
c~
a.
in ~ U Y
o. ~ Q IZ J
7
W
UO
U
~ Z ~ Q Q
Z Z 4 t- CC CC
(O ('~~7 O CV N
a a_.
Z n N O r r
U ~ W I-~- 1~-
p ~ CL
U w Y Fm-- n. od~C
a
m az °~~ m m
- z ~ a. >
r ~ ~ cn cn
_ E H ~ ~ Q Q ~ X X
E=~ ~'''mz o. OO
m a N U ~ Q ~ E- I
Q .r tO Z c uJ W Y c o g
O O ~ .~ O ~ O Z .U .= Z Z
~ Z = p- ~ N J N = O y J J
m r O H d 0 = c LL CD fn uEf tNn N F- F
U w
fL d 0 ~ O ~ ~io U r p ~ ~ a ~ F~- m m r c
N UJ a ~ ~ O > ~ y z a p o t c0 O C~ M C ~~ r
~~w0 c ~a~' ma. c~'o~p U ~'' -°'-~U U ~~n -''
H fL 'U ~c J ~r a>Z cZ~ ~ o. E
V E Z ~ o U ~ U Z ~ Lu °' of ~ t .S r ~ ~ ~ ~ ~ E
Q C7 r -R p a ~~c, ~ d a. U '° c~ cn a~ ?? ~ c E cn H- H- o v c
1~1J NO1- Z UO Z~ cO CH'Z VZIQ- N O~~ p ~ U~ O
F- c0 LlJ Z N ~ d I '_.. O ~fy L ~ N = ~ ~ ~ r V
Z C~ J ~L N O = fn ~ a U N ~ '_. Q Q C ~ ' O ~~I1 U7 ~~/1 0_. LL E
d. ~,J~~=~~, rMO Ua ~~~(naN~ ~~ E 0-UV/Nv/ m N
-i c, E- -W~ n. ~y Z c~ ~ m U °° u.l a~ ~n g o Q U Q ~ o ~
Wloyr~~z~s~ Ea. ~ul cp~~p~~ ~ ~ °~ ~IfoCf--cL ~~Wo
cWNa=aaz~c.~~ cc»cn =c~n.~c~
v
Y
Clf7~lc~~ ~-tON~~ tOt0~00fr-W t~000Nmr'00~ I~NOOtOIw
r O i Q O tD tn P71 N O O f~ 1n 1~ 01 t~7 Cf r ~ ~ N ~? O O ~ ~ O ~ C~ N N ~'
N
dp O, N7 ~'' tD Q ~' ~1 ~D f~ N c~ 1C1 ~ N f~ tn Q ~ O h O O p~ ~ ~ O,~f~~ N
CD CO OMI C1
C~IffOt01'~ N10N~Nt~ ~-OO~N11ff11f7c~ c0'raM rM~~yM~p ~p~O~IN~-
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J~~x°~'~~~~~~xxx~x
L a
i
.I
- ~ 17-
CA 02290738 1999-11-19
WO 98/53103 PCT/US98/10561
CABLE 7 (CONT)
Q _ Z
- d r O N I~ O tn d
~ I~1 Z D U O ~ z Z Z Z Z
c~_ngN a~wQ Q QwZ Q ww
H w C~ cn F- C~ U J O F- J O O
ul O J cn Q J J ~ CC O 2 CL
E..~-..tea c~n~ a' can cwn Q~aa cwn a
U w Q U ~ Q Q Z Y z ft Z Z Z =
Y Z CO Y Y Y ~ ~ ~ Y tn (O ~
z Q z ~ ~ O ~ w w o ~ n. w O O o
Ow~ zo c~ z z zzr~ z
~ a
Z uJ.l ~ ~ N ~ O ~ ~ -~ ~ ~ ~ r U
Qga
o~ ~Qn~ ~ r
p a = z p z i: p ~ O O m N Q ~ N N a
~ f-- ~- z
U H p n. N. ~ U ~ ~ ~ ~ ~ U Y ~ U U T
n- Z Ct ~ V p ul U ' U U ~ ~ >- U
m iL h- ~ ~ ~ LL J C w l1J (n ~ C'j ~ CL
a. Q ~ w pC a p aC Y 'd ~ ac z Q p ~ p
Z °~ D ~ (n D J o D D Q fn J D Cn fn N
m ~ ~ O w ac rt
O ~ U ~ ~ O = ~ ~ v ~ ~ ~ U O ~ U U W
o U = la-~ m ~ t'ii ~ ~ ~ c ~ ~ a~. d = ~ ~ a ~ ~ U
m .~ = CL . ~ Z ~ 'a~ r Z yn u~l a .JL' r Z N ~ w
d U rn a a ~ CL ~ ~ H.. CL CL tn
z ow~Z~p .w~ ~w ~(CO.- . =ONO aQ
w J o~QUW--YCCa.?a~ a~p~Y~ ~ ,.-=r- zZ
m H - ~ Q- I1J F- 0- I- O O O UJ ~ 2 ~ O Q ~ 0. ~ Y Y
C9 cpt -D O °' ~ H ~ U Z ~ Z ~ ~ ~ ~ U w I ~ Q U w U Z ~
O ~ ~- C9 E ~ Q a- ~ p ~ Q '~, ~ ~ g o~C O ~ U ~ ~ O ~ E.~.. V
f- 1 U w p ~ w w w m n~. w J ~ w -- w ~ Q ~ Q w ~ o? ~ m O U
U ~ a m ~a ~ oC ct ~- ~ 2 m w z ~ Y w w w
Q ~ E a ~ ~ aw. U ~_ ~ ~ a ~ n. a ~ U Y n- ~- n. U ~ ~ w
~~.C CtIC~O ~D~LLJ ~ ~~a c~LlJ~-L1J~ NI]-LlIW
1- Z CL ~ Q ~ Z E- Q CO O Q N m H ~ z r CO F_ ~~f"" Z a.
z,N ~'"'_ _=zr zmzN z~ ~ z~zw~~,~zNz_Wz_v-~nO
+ Z w j ~ v ~ Y Q ~ In ~ Y ~ ~ Y ~p ~ YIN ~ Q o ~ Y ~ ~IfL ~O ~
JIv a °~~ f-H- '~=tea=Q=~ '°=OC > =~=Z~ >=w=z=~°Cm
ILI~. IJDI~UIIJ j~~ JJ~n.z~ J ~N-~ c!5 4-JN.-~- ~~- Jd
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_
a.I ~ ~ ~ I N
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c ; . _ ,~, o l N o ,n ~ <o cn r~ ~ cc w r- ~ co a~ Q,
(p . ~ C~l i ~ py O~ ~ c'~ O O t0 O ~ ~ O N lf! M C~ f~
m i n r O Os ~ h cD M h C' c~1 ~ I ~ ? CI h c0 Q~
O Ins h I,f~ Os r ~D O N f0 In 00 GO O ~
Of ~ ~ ~ y O tn O O ~ N (L !~ fD CW - c'~ c!' ~t
~(~_ ~;~ ~ S~ I~ > >~~ Q > > IX IN J J J
i i
I ~ I
-1 18-
CA 02290738 1999-11-19
WO 98/53103 PCT/US98/10561
TAE3LE ~ (CONT)
I. z E cc
w ac
I
O
I
=
I w oC U
O
U a>' Q
Z
Z ~ ~ u1
O
~ J O _ Q
Z Uf 1
ww c9 = ~ z_~_
n~. ~ oC ~ Y
O Z
O z _ O ~
a
w n z
a Y a
w O Y
~ a z d ~ z~
rw ~ ~
,-
cviQ ~ tt U r c
Q CL a>
O t- m.- v.
-
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,c
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~ U o Q
m w ~ ~ ~ wo
O Q c c_ c
w w . . ' a
F-O O ~ oN a a o
Q z- Z d ~Z c ~Q
~ ~ ~ ~ w ~w
~~ a w U~ ~N ~ Q
ww a-I- J c ~ a
m
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~ ~
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a ~ u n
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~ w p Z U 4 U ' U 'vE Z Y~ ~ co
w ~ o ~ ~ w
U w ~ O U c a~ U
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.
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w J - ~ l1J M O Z
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CL Q w0 c ~ ~n v C v, 1'-
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I ~ ~ ~
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la ~ ~ a ,t ~ w
z ~ ~
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~n
I
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a
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C~,~ Of OlON r O OD1010h P1OftnI~01 <DN~'QfIO M 10
10~. tn t'~N CD~ O1 C00 1t70 r('~OfN W ODrl7N Ir~N p~t0
. C1 CDC1D~'~ t0 InN <DQIrr rtnO 1~~r CD IA
m M t~!OOftt r ~!OC1N Inf~!'~t7CI C~1~M I 47
M Q1 ID
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C3 ~~ ~~ I~~ ~~ 77 >> >l>>> > WI~ IX~X.X ~ X
~
I
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-119-
CA 02290738 1999-11-19
WO 98/53103 PCT/US98/10561
C'ytokine and C'ytvkine Receptor Arruy
In the cytokine and cytokine receptor array according to the subject
invention, all of
the unique polynucleotide probe compositions correspond to genes that express
cytokines or
cytokine receptors. In a specific cytokine and cytokine receptor array of
interest, the spots
are as provided in Table 8.
_I
CA 02290738 1999-11-19
WO 98/53103 PCT/US98/10561
TABLE 8
I I ~ I i
i I
I I
i ~ ~ I
I
,
I
' ~ ~ O I I I
o (~ ' ~ I ~ i Z
o u. . Q C~ ~ cu Z_ ~ I I Z
v o ~ LL Z Z U ~- ~ U U7 I ' U
o u~mU ga m I g U I a
a ou.m ~c.~ ~ ~ I z
U U Z ~ Z N a~ ~ p Q
~0 E W
t _U Z N ~ ~' Q J
>-~ o H U O
o~ CG ~ IZ-. O O ~ ~ ~ ~ ~ O
O ~ ~ J ~ >- m ._~ a (~ n.
E a ~- o j O .~ O ac ~ rt O V w
dU O~uUI QV ~~ o ~ Z p
to o ;D ~ ~ U a 1- p_ = Q ,~ Q p J ~ H
Uwz tZUv ~~ -~ ZU ~ O C'3m
_oo ~ cn o oa Nwa
Z Z_ ~ u~ 0 '° V ate. a U a0-.. ~ J J ~ y Z l- ~ a d aZ.
- to c F- E N U J c a a Z Z Q ~ a
d d N ~ o ~ N ~[ m J a LlJ ~ Z ~ O ~ n. F- I- ~ Q Z
U U p o ~ ~ ~ u. ~ ~ u. u. U U o U Z '- Z J uJ C'3 uJ ~ Q
a a a c tU1! Z ~ ~ ~ ~ Z U fn a ~ _ ~ ~ CC ~ H ~ p p V
V nCC s-Q Uoa .~~~ cnOZ ~aJltw'Z OO= OyOYyf~ 1-d-
~ CC ~ _ _ U UU U I t~ w
d a ~ ~ O O ~ ~ a _ ~ ~ (.~ O~ ~ m ~ o a~G~ t~ a ~ a ~~~ a,0 w
! > U U ~ ~ - O U U O ~ Ct J li = = Z 21N =~U~
Qa~ O~ ~I-a ~ZO OtLp U.f-O F-'j-F-az O
O IO a ~o~~ r-mOZ aCw~ ~ i."' E-U J O~cC ~'Z~~,'.'l a
a o~_. Y >==z Q~-~~ aF-a-UQ= Uc~aa~wlm= ~Ot~ 0;00=I pu
uW a ~ m ~ ~ O ~ N o ~ a Wu n- ~ uWt ~' a U~ a a Q ~ ~ ~i0 ~I~IUI uV.~
w~ ~, Z X000 ~ZjnJ. Qmn~.~0 n~.wina~i'~z~i ~c~~ c710c~o~~loa~
~Q ~ cn~eCL~U UItJ J aCLvcomO cOI~ mW /LEI CC rcu
n(~nNC~p ~C7C7~ O~CLV r' ~'?~C~ ' ZCCZ l ~ ~',ZZ ZI~ZC7IaZZ
ZZZZZ~ ~uItiJC9 VOQd ZZZ= JZZOU Z~Z IU YY
_Y_Y__a ~ CO YYY~JYY~~OYYOOUQH CL'
d Y O Y Y Y ~ wl p- ~ _ _ _ ~ > > ~ ~I~ ~ O p 7- O'OIOI J
Z lL LL LL ~ I z L1J i1J LU J ~y, (] LL :.W LL l ~ I Q I ~ lil
E ~~»~Z__m a u~ILUIQU t~ O I
ZZ J JJJ JJ l~ JIJ LL>(/~ Cn I
J l1. JIJ J ~ _. - O ~ ~ O ~. CL: _ ~ ~ p CL Z p :ZlzdillZ',~ICC
Z ~ o J J Z ul u. ~ ~ (L ft ~ w,0 d !- Z Y Z O'umtulul
d wl.~uwlwu~,iO~>>U ~o~-C7 mmUQmu.~~:~guyu.lVmaa= Qa'a
~I,.-~-~-y-.I-~ cncna ~zgm--~-,.-a~zzZ~~z:zaz~~m ~~~ccin.,~.
V - - -Z,Z ZpZIZ~a y Z Z '~ F- Ct1 IL a z Z Z ly '... I- J -,- 11 - U' I- J iF-
U' F-illl!=iZiZ
i I ~ I I II!l
!
~ ;,I
I .~ I ~ ~ ~ ~~ ,
C O N CO 1~ ~ O V ~ M O ~' V' ~ tn ~ Q r ODi,O N107 ~ OIOD ~ N N Nl0 O~I~
M!~,~j~l~ O
m O I (O C~ Q cD O ~ ~S f0 N I N ~, 41 i ~ i ~ i~ t0
(DO ~~~M (D M~,,I~~'NOf~Qts yDVM_N OMODrm,~O~NM_V.f~
~~N Ilp N p M cl0 X O O ~ I~O CIO) NIO N IOIp O~.O OI IO:OI
c9,~,x ~,xIQ ~ Ixl~ x ~ ~x ~.~ x Y ~ ~Ix x,o ~.x ~ DIY ~ x x ~:x!-~,
;~I~ i
;, ~ ~ ~ I I
~ I I ,
-121-
CA 02290738 1999-11-19
WO 98/53103 PCT/US98/10561
TABLE 8 (CONT)
i i ,
i I
i I
I
Ir ,
I i
I
i E U - U Z o ~'U'
uJ (O w w ~ n
ar v~ ~ i CL (' C
O ~ O I O
m ~ ~ U ~ ~ a
'1 N a
N U UUJtJi. W~Z t~
Q ~~O ~U
o llJ
a a p e'' ~ N w z
cL U o CL
cr U t4 ~ U O w t- °-'
cL O O
Z ._ .c. a c ~ ~ V d ~ ~ Z ~
Z ~ ~ Z o ~ ~ ~ U w U Q a
U = Z a a L w w a ~ a ~ O Z M ~e fL
a a ~ Z ... a U .- Z Y CL O = d d
ZZZmwU ~a E- ~~ OY ~- Ot-Z~ .ACC Ow
aQa za a= ga ~ f-z
= Z S Y ~ w ~ v E- O ac Q w ~ Q cij w p ~ ~ w O
U U U ~ O ~ U Q ~ m t- 0 ~y F- ~ ti Z U h- w U ~-
zzz~p~ ~ oac ~w ~~ wlw p=~ww w ~wo~~
J~~ a w ~ z ~ ~ ~~ ~ _ ~ ~ ~ a,~~ ~ ~ a a ii a ~
alaa~~p U~~ ~Q ~ ~z ~cc ~O~zwa~ U~ oc=~o
I ~ ~U~ aw~. oti ~~tu p(n ~ul~~Ur 'L(nw w~ IU~~~h
O aCO1ct01~ a O U w ua~ U = O ~ OOC a Q pOC ~IQ 4 w Q cr ~ Q Q Q ~~ z t~u w
E- -:I- II- Z U Z_ D Z Z Z u. I Y CL U
t~matJltat~lm Z ~ IO ~ a ~ ~ p ~ w V w Y w V Y V ~ w _w Z z ~Z ZIP CC
U U Uli O ~ a O ~ z a. ~- o~ U w ~ z ~ ci Z w z w wlrz- N z W uu ~ IWu ~ w
mm a,oCa w~~_mn.c'3wa~,.,~w -~a~uu~CZO~ I»a~
2~cLJww Ui--amUmO~oZ~ O~ __~ QIDwOf--
c~»cnau.U u,~OC7~-wUZa>r O ~ D-I00 UO 02 I~OpZ
~ O~tl1_ ~ ZO OZ~wmIZltwZ~ ~C'3 ;U~Z
ZZZ'ZLU~ ~OZ~zCC~ZZ~a wj ~~In.l- >Q~ n. J a4. Y
d Y!YYIOI- ZwO~ z~YYZ~ CL-- OZ'.u1 w~ CCIw IuJQ ~ul~tlJ
E »mr~zw wzo~pz_p»wz a wzwc acvwz z~.-. u~zwz
tp -wwlwiw -z C'~~w~OmLL J JLL- ~-~ ~ mE-[[~IJ.-LL- -~ 'J(Olu-(O
Z J~J:J~.Il a,Y O ~ _ ~ ~ fn a (n ~ ~ ~ p (n (nl~ ~ ~ (O CO ~ m w ICL
~;CLICLICL ~ ~ ~ ~ U O Z ~ ~ O IU - O O a.IZ',U ~ O O ~-. O O
yulmlm:wlg~~ p'Q w ~ J rr a w w ~ cc Icn ~'. ct c~ ~ Jia v ~ ~ o~ U ~ ~ ~ a ~n-
w-I~
~ ZI=~ H CL Q12~~ Z z O >- a ~ = Z.>- ~ICCIa ~ Z 7- ~ >- 4_ ~ d,l-Izl
c~,z;zjz;zlC'3 U a U z w UIH I- JII- i> wi F-la I- u.h-~> cvl_ 1- _ I- u7
i : ~ . i
~ ,
I
i : I I _ I i,
C NI~IV_IN tf71C0 N O ("7 O h ~ ~ n N ~ ~ O M ~ ~ In 01 ~ f~ h~~ ~ O p N (O O
m fD NIO OICD 'C
N N I tn I CO _ ~ m Q1 t~ 00 117 N O (D CD _ _
OI ~ N _ C V r c~O ~ N ~ O~ O O an0 O~aD,.~IN_
O C~ O N ~ O ~_ O 00 ~"~ _
C IQiN u7 ~ N O C7 OI .1~ mI c0 O anD~CO ~IQ7 (D m ~ O (~ O O O a ~O~
I ~ N 1~ (O tD , G7 O (D fD Q . O
I 1 ,
C9I~~X~~;XIXIO ~ ~ ~ ~~~I~I~ ~ ~ ~~X ~ X x ~I~~~ ~ ~'.~:J._.~ X ~ X Y x X ~
O'~~a
I m
:Ivi I,I II I : ;
'~ II, ~'i ! i '~ ~ t
-122-
CA 02290738 1999-11-19
WO 98/53103 PCT/US98/10561
TABLE H (CONT)
I I i
I I
I I
I I
W J _ I
! zJ
U
J W
M
~ W W ~ Z O
O
~o ° ~ g>
I O ro a~ a ~W N
O N lZlJ N Z
H
Q V z C_ O (n
C LU ~ Y Q Z = tin
u- ~ ~ ca ~ f- ~ Z O Q c
I ~ _~ Q ~Q Q~ ~~'m
d O O ~ U U = 11 W~.- O Q m
O a a W W Z N' u. o ~ W~.- O O U
u~1 ~WUU f~i-a- a~ la p U~ U~.a o,
U O Q W W '' CL fn Q W N ~ W L ~ V W W c0
CC CL Z m Y O Z ~ 0
_ U Y O O a ~ a Y '' I- I-W- O~- + ~ ~ fL o
~ LL 'W J J ~ ? m z w ~ ~ U O
E- OC7Z»mHO" ~f' O~~ am ~W t- - ~ OO'~ N
U . t- Z cn O O O U ~ ~ O O p, .r Z Q m U .c ~ t0lJ V U U r
Q ~ a W ~ ~ V V a ~ Q z ~ d U Y Q p m uQ. m ~ I~~Q Q z Z
1- _ ~ U ~ ~ ZIZ W W W ~ W W W W O = c fr ~ UILL t1.lW W
W - -W ~ !-- w U O I- t1J f- c ~ I ~ O W F- 1-
~I~ Z H 1-W- ~ V ~ ~ ~ O W O ~Q ~ ~ of ~
pctH,y00Qa Hcn o~m ~u-O ~~ Win.
~ ~ O ~ ~ ~ a n~. Z_ W O U ~ ~ Z a O O miU oC ~ U D O ~ O O U U
>- O Z m - w o O W Z told ~
QJ~QZaC~C7Yh-p~ ~~ YU ~~ n~ J CCQ=~ UIC7C)~ij t~iJ
u.QUu.O W WO~DZ W g Z W UU m0 Q ~'~Nmi-idOZ Z
Z~W~ CCJJ~OW W Zu- tL~ W W UCC ~ QvZJ OZIWW W W
O O m m W Q > C'3 W Z H W ~ CL M O ~ J ~ ~ ~ ~ W ? > p p
~prOw~mm~0oc0 C7W~,OQ .-mr ~ ~'_' ~ W~ctl,ct0 O
Q z ~ W O O W W Z O p Z ~ Z z z Z D D = Z a Z O p U O O D ~ a
~~YIUQU~CLU~m~ ZQJaY .=tJYZZW ZZ ZYaU~Lt~~
W = W 0. ~ U = W ~ W W ~ Q Q Z _J J ~ O CL a I- h-
Z I~ ~I~ a~ O N w ~ O ~ O O I=- in uw W ~ H -r J J ~ ~ t~7 C=7 ~ ~ p p Z
u~~lw~un~ ~
d~ ml~lO~tp c a~v_QHW Ha~OOr-NOO~onQoOW W ~Ov-t--~~-IE-1-W W
c;alm~Wl~ U m n ~ d N. z O U D Q O Q D O W ~rIN - M U aC CL CC U U Z Q Q Z Z
d JW,~' > Q ~ ~ ~ ~ W W O ~ Q Z >- OC z~Z Z f"' D OI~ G W W W ~' = Q Q ~ Q~J
JIO O
C7 IC7jWlzlt-~~ W =I= = Z =Im W ~ W r- d YiWIW Z U UIU UI~ I Z Z F- ti ti
zl~ia. d.,m m
, '
I , __- _ _ W n -~ r;vc_naooo~
INIONIyCtf~Or7 V ~N_ ' OI~pO p> NM '_~OInMONO V'N~N~ n 00 O
I~IOQf,OiMMNNN MIfIQ~M~pM NNO Qll~~fD~.,~_fDd'i17000 I M
;~~~efiN~nO)Q~MOQnN~~.,~r-Or tDCOR7QOr~NNQN~cOO~r-OIrvfl.fDl MpN
tr W Dln nIMInIr O_ O_ O Q fD O N~M1~ ~I(p tD tD COIn CDICO IN ~ _ tn !N~NiNIN
~ N
41 COIO~NIMIMIr r4r r M ~,O~O OIO 1010 O O OI,O10~ O I IM J j I
CJ I~~J,~;~Ir2.~ O D DIO D D r..-7 M M:J IJ.J J J J,JIJ~J ~ ~,.W JIJ ,_.~
~I~,~,~~:rL
j
! I I ; ~ ~ 'I
I ( I. I
1 I ~ I ~ I I !
! II
_~~3_
CA 02290738 1999-11-19
WO 98/53103 I'CT/US98/1O5b1
TAE3LE ~ (C.ONT)
! I I ! I
p ~ ~ I
I
I
I ~
I
1
U N ° Of- i I
I m - u~ E.. ui ° n.
-- I j uJ H ~ !
I ~ m ~ V a !
i Q ~uU.~~ c°~Ou.~ I Z~ m0 °vu~'
f > 5 m ~ ~ Q m ~ O ~ m
s=zZ ~ ~o ,~ ~ o
~~y/ N ~ O J t m ~ Q U ,:a fa
o_> > Z o CL li, ~ ~ m Z m ~ Q Q
m ~ ~ c U ~ a ~ ~ ~ Z ~~
O ~O O mmt~Q -C3Um a =~ ~ >
c~ ~ m n ~ C9 a ~, ~ ~ Z ~ a O ~ E
Ca'J °~' ~- v JQ tcv 0 ~ ~ uU,J ° U ~ N in ~
~ Z ~ w Z ~ ~ ~ ~ Z a m cn ° ~ m
c _
a ° o O~. Cyi ~ m Q = r .-, O CL m w CL ~O ~
-' LOH.. Z~d~c~ EZ Wi _°H- QQ ~ ~d = °' du.
_ IL m U
M ~ am. Q O m ~ U V Z Q ~ m o~ ~ U U U a m x Q
v
c ~ ~ n. U U ~ cu " Z a Z tin = °? m ~ ~ O O ti w ~ '' '° Q
~ r o ~ Z J Q V ~ ~ ttl ~ -Q L_lJ ° ~ o C7 = tUlJ U U Z ~ ~ is ~ _
'~ O~ ~ L ? Z_ V = ua.. ~ Z = U m ~ p w :c ~ O ~ ~ ~ u- ~ ~ z a. Q
Iza1-~ ~mm= = U a Q~Z" CCH-~~ QZOm = UQQI
m O ~CCD .I-=QYJ 1-~ZOUO v,~r
~ >- m o ~y Q Z Q Z It w Q m m I- Q ~ ~"' ~!C7 U ~ ~ ~ ~1z O ~ZIZ ~~!~Q
O~~a ~ EF--(~u.~ =zCt m~QUu.n' c-J~~U It ~
o ~Ow ~, O O
cUmC7>mcn mti mO~~C7~ °'cn~Y~ mmCL~wu.! ZU ~IOQ
cQOJ Q O O~ Zf.-IO~..Y~ C~ ~ = U f
UIg UI ° ~° ~ ~ a m U t1~ O ~ m E- ~ = U ~ w ,_ Z u. = w T O
u~_. U Q = iln~. a'
E-~Q d s~=UJUY-~CC>-= n.p~~yQ~~QUUF- ~ ~°Z! F-0.~y~w
u.I H v f' Q w U C7 !- Itl U ~ U m J t~ ~ ., - U
Z ~ O Z v' ~ ti. _ ~ J ~ h- LiJ '_' U IL Q U ~ ~ fL LI! ~ Q >- til = X t1J J U
O ~ ~ a Z 0 ~ ~ a ct ~ ~ ~ ~ 'n ~ ~ z ~ Z O ~p z ~ ~ m N ~ o~ ~
p ~ = z ~ Z ~ O z ). ~ m m tn = op ~ Z = ~ d ~ '' ~ Q ° ~ F- ~Z-. V ~ O
~ N ~ ~ ~ ~ Z
Z U ° ~ ~ H cn (L ao Z = ~ Z ~ Z w > CL >- Z - - a -
a C7 Z ° ~ U to _ Z J O ~ ~ ~ ~ ~ ~ U Y V J m r O ~ ~ ~ U I ~ YIYIiu.Y
°
Q LIJ ~ ° Y J - ~ ~ Y t1J Q
E OI= ~ O Q ~ u.l oC m Q m ~ a ° O J -~ O ° O J U a umu n.
m > >IUI° w
~ I- ~ Z g Q ~ - N ~ ~ uJ m p m O 2 m J:~ Z p Q Y = ° a~= ~!~~~i~ Q
U (n~ZIL ~NZILr. ~JI-UQ~a.J~iU~I!-~ ~I I ZI--~Ji~JO
d luu~ Z;aQC O = j o V ~ m V V ~ 7 ~ ~w ~ ~ ~ ~ ~ w a~w aid ~~~ O;af~ Qio wuu
~'i'~=
e~, Ipla Olw z ~ ~ ~ a w "- g Q ~ °° O H ~ O ~ ° -' ~- my-
- w!m w~c~ mI~I° ~mZ;~-- i--!Q~t-~
Cf ;m;~;~':Z O Q z r- > a ~ a m wi = u- Iz a ~ ~ O,1- z U ~I2i= _,~ c~imlC7
U;m!z ziu..z Z
i i
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y ~~ I I .-, . _ . - . ~ I ~ I
C ~ Nl~flil~ OD c W !1 f~ 0~1~ N ~ 1v O ~ (D'O O O cD N.tf~ O~V CD~CD W D CD N
~i0I0 V!N N
OD C V ~O cC :(O f~l~ N I~ O,OiQfl(rIO1 ~I('~ OD
Q7 I ~ . Q' fD m O ~ fD 1~ ~- N CO m ~ V ! c'7 O N I _
C~IVI~ICD N~f~ ~~~- O) ~ ~t 1~ 1~ ~ M C',~ C.,~ M~1~ h N N ~ I~ QWQf O!.O 10 r
NIN,tnlufWnllvifp C'J
C .Nic'~Ie~tf7 1~ Oi~ ~ N N ~ c0 I~ ~, O
pl : N N , N ~ N N C~ f~ C~ C~ c"7 M ("~ O f0 (D (~ tO tf7 WIcI7 I ~l'71 ctf1
W ct7 C(D ,~ ccD (D (D tD (D : (D i CO (D ~ CO I CD 1~
I l I I I i
I I I i ~ I
I -i I I I
I
I I ~ ~I ! r I I
CA 02290738 1999-11-19
WO 98153103 PCT/US98/10561
TABLE 8 (CONT)
_I II
I
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2
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oQC +~ uJ.z ~ a r~ U . m~
_ C7 ~ C'3 U ~ Y Z ~ U r~ Q ~ ._. ~ a~
h J u. J ~ T. z C_7 ~ ~ N Y j Z ~ ~ O
~ h w L- 'p ll~l T a u- ~ w
w ~_ w O V' ~ Q ~ N N ~ li J ~ ~ w
N O_ ~ O N j h ~ ~ V ~ ~ m U' ~ am p f1
O ~ O a c0 (] J w V ' ~ ~ .hJ c0 V V
tv h w Y w u. z D .- ~ CJ Y ? li ° t co .:°_ O
CL u. ~ c n. OI- O U .~ o ~ m O a ~ ~ ° li 'g Q
.E ~ O .~ O w ~ t" n c w O~C ~ Z ~ ° c E ~ d w
.in Q ~ Q ~ y ~ W N cn cco m ~ ~ C~'3 lUtl z ~ .c .c a °~ H
g
~ Ow Z C7 Z U o U u1 ~_ w ~ ... , ~ ~ a O 3 ° o ~~ U
.- c? " ~ Q n Q '° o D ~ d ~ U ~ o '
f~ O O O O U ~ Z N Z E E' Z J N O ~ ~ - C V ~ h
V O ~ a_ ~ n~_. ~' O Y U Y d .c C~7 O ~ a O ~ a~ a .S Q ~~
E d O ~ O CL Y h Z tL w ~ V J O. ~ V V 1w- ° c ~ .c >'
w ~DU_~U~QttI~Z d ~ N Zww ~~ ~~~~;° Z
ww ~>~> ~wOO~ ~ ~ ~ U~ w N ° E~ O
Y OC U ~ ~ ft ~ _ ~ N O ° °c ti Q u1 ~ _ ~ N ~u .~ U O
'yu ua..wUwUY~a=~ o ° c~~ >~~ ~ ~ a ~ytC U
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hQ Q fL ZwJ V UOQ ~~ ~ =h U
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(.~tilF't-F-hU1'.'UO~tLfLl-.-Z uZ vd p ~wZJ~ZZ~iZ_ I V
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m ~OZ~.r-y--.~u~°~.,x~» ~,~ ~,Y w~zQ ~f-QU~OZZ~w~~IOL
~ aC ~'U "~ "'~ z g N O ~ w w > w _> O Z m ~ ~ ~ Z J ~ a ~ ~ Jlw J~~
Z h ~ O~~ r O N O ~ Z ~- ~ tall ~ ~R U ~c° ~ Q O Q Y ~I> U O h- C'31C7
!~-i ~~ZQ Z
y Q --BIZ ti H-- 11. H- O U ~ ~ U E E U O ~ ~ ~ w~l-,cn D J;.J Q ~I Q
c a J r Z C~ U C7 U CC tn °- U U Y
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_ p5_
CA 02290738 1999-11-19
WO 98/53103 PCT/US98/10561
TABLE 8 (CONT)
j
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LL U
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m Lu ..' ~ ~ o D r, ~ CL
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0 U m a ~ w ~ ~ ~ t Q ~ w ~ u' a
f" _ ~ tC O r a Z ° t1I
V cL n~. v M ~ Y a a ~ O Z Z v w ~
w w w N ~ ~ V ano = ~ a Q ~ CC Y ° a I ~ ~_ ~ W Ct
O. Q ~ N U ~ Z ~ CC m > loll a ~ ~ ~ a p X O Q p
~ O D to ~ u1 c ~ ~ J u1 I- B ~ w U ~ U Y ~ Z
U maws Uz ~ w~ DwoOC ~~m ~Z~m a
U Q j o. r ~ O a ~ ? U O~ c tly a. ~ p CC O t~ = z Z Y to Z
a f-- U ~- ~ ~ ~ ~ O ~~ tL U > X OC u.l ° ~ ~ ~ f- 1- In O V
u-~ w 'rtmHU C~~ ~ ~a~~ ~OZYCL~a ~O~O Z u:l O
m N O O Q Z ~ a~ m u- a t-- ~ O w U m O ~ ~ ~ Z
H ~ u. cn U O = U n.
u. M U a U O = tn CJ o a _ M a ~ ~y ~ m -o U t- Z Z Z
U' ~ F- llJ a Z Z ~ ts_ - C7 CL a -'~ LL ttJ CC o
O ti Z_ Z a ~ J l11 c z Y ~ w Z O LL ~ Y !- tL
= N gI= V O U ~ > M E Z ~ _ ~ ~' ul a ~ ~ w U ~ z O Z OIQ ~ E
Qr ~ ~ (~ lL 111 ~ UJ ~ ~ W C'J U IL =tt Z ~ g ~ I- u. ~ ~ z U ~ a Z =
Z O Q O Z ~ ~ C7 ~ Z U 1- O a O ul i- u1 ~ > Q p 2 O w ~ Z 1- U to
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o w ac ~ cc a ~ O ~ .-.a H cn O z o °~ z =,O z
Z U O U U Z ~ O ~ D Z a Q Z H Z r a - u~
a ~ a a U - ~ Z ft Z a ~ ~'- w U U cn O to tn Z Z ~ C~ ~ N D n. CC ~ Z
d mu. >~=a~cw0~=aY ~~oCU_,zYZ_z p~~Y an. ~-til=z-~IU QH
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Z (L ~IZ OwJOQU~JJn.cntnz cmUOrU~rQ~~a_ZZiLCOXZZa~ J=
ar a QI mU~~>r'-'E'-'om~tOZmw-Of-~~ ~ H- O~O lm
aO~p?OU w~1-~CC~vwcOaCa °'OE-O J~ °J~'=YO--o~gz-mO.JO
c~ =~miz~~g?°a~zoZ~l~~'z ~aa~N~lZ ~.o°a.=~~~cYn~~~~i?I~?al~a
I I _I t !
~c j I ~ ' l
C rtDll~Mm(DON VItOMn07nr V COMMtDOIfi OfNr a_0010W 7N~ ~O~Mlff
V M ~f7 n Q1 M n m O OD N c0 OD I ~ fD In
m Oc'7n(O~n107ntfl McL707n1n~N(D ONNn~OD O t>.v NN~IO~NO, ~COCD
C rrlf7IMMM~SOf O NI~OOOnMM NtnMtl)~Q ~_NQ ~~InOOM'rMfDln
CI ~L7 (D tfi vff W fi CO 1~ n n O n '- O ~ O M M ~ I fV I M ~ O 00
X X ~ XIX X X X X X r N O -~ ~ IJ J ~ ~;~ '~ .r,X XIX XIa,O ~I~ .-I~ ~
C5 X ,-.
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-126-
CA 02290738 1999-11-19
WO 98/53103 PCT/US98/10561
TAE3LE 8 (CONT)
I
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C
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X~~z~ Ya + ~-~'z~ oQ~aQ ~ d ~~ v
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Q a Q ~ '~ -~ ~ m u! ~ Z Y ~ U ~ ~ Q aC u- n~ m m m ~ m .~ a~ ~N.. ~i u- -; m
I Lll J O ~ d' U U W CL ~ Z SL LL u. Z N iy ~y Z U N ~' C~
c~ ~ I~~i~Z~ Zzz ~~Zi~~~zmi ro-~~v~~~?zm ~I~I~~Imn~
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J N
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I
_~'77_
CA 02290738 1999-11-19
WO 98/53103 PCTlUS98/10561
TABLE 8 (CONT)
~_
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m
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o ~ zzzzz Q.EL QzQ m z-~.OQ
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o z z ~ ~ o m ?I~ o p plp z c~ o c~ c~ p rz a N ~ m a m;wlYlc~ a c~
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~ > >I> > > X X Q Q > > ~~~ J ~ ~I~.~ ~~~ J ~W ~~ ~ ~ ~ S ~ ~ ?.XIXIX
I
I I
CA 02290738 1999-11-19
WO 98/53103 PCT/US98/10561
TABLE H (CONT)
N h O ~ ~ I
I p U O O z Z Z I'Z Z Z
a ~ ~ a a a ul Z a 'u~ u, ul = m
C7 vW - C~ C7 H' E- ~ ~ u..
J fn O J J J . ~ O Q ~ CL O m
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a U w a a Z Y Z ~ Z uJ w 2 Z t-
O Z Z o
Z Z Z_ Z Z Y ~ ~- y ~ tn ca ~ U
Y cn Y Y ~QOa to O O a O
Z ~ ~ Z Z Z Z ~ 111 Z CC
tn ? (n fn ~ ~ LL O C. ~ a
IL J r' ~ ~ r r Y u.
!- E- t- Z ~_ ~ _ ~_ Z . ti U
W
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aa~.~ U a~. a~. ~~UY w U U T, UYO O
w w m uJ
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tL 0 CL CL CL Za p O ~ ~ I- O O N O V V
rf7 v D tn O ~ D D ~ tn a O fn fn (n N fn
7 Ltl ~ fL lL 111 LL ~ O LU ~ CL ~ J t1!
F- CL ~ Z = = U p a ~
Q ~ g O V g ~ g g ~ U O ~ g ~ U U ~ U J ~- Q ca
u. y n. ,.,~ O O u~ Z u.1 . uJ u.l to m O
~m~L~1! ~ s~ ~ ~a'Z2 ~c~~ .~~E..~ V a~0
m c ~ ~ ~ a n. 5 5 = .~ Z w = . f- uW-
i~ .~ i~ i i ~nw I '~'~ a.-ZN ~NrZU JQ
Q .O [~ J ~ Q lL UJ LLJ LL ~ ~ ~.~ I O ° ~ _ ([ O N ~ ~ a O ~ ll m
U a
oa'u~~o°.. o ou°u=Y ! ~ ~ooa°~-~a zYa~z
tn~OwZ<nQ cn cnf--~Lll~ m mcnZtt~wttjw Y Julul~ . oIZ~' U
~o ~°~''.°~wmma ~ ~gwmu=, ~ ~ ~?a~,.~,,ocw z,~wa~~°'
X ~ Q a ~ O ~ U w ~ O ~ D ~n ~n U U Cc CL O Ct ~ U ~ O~ ~ X,w o
'o ~li ~~caatUt~~ w ~u~~QaQ v~n~_o._pwJ'"m~m OU'Ln~".4UOU_0 ~~U ° o
m E n~. ~ U ltl ttt a Y ca per" ~ a ~ a U Y ° o o Q' I"' .Z'j ~ V d N-
~ j d U Q ~ p ~ ci E
~ 4. U J >
d ar ~_ 111 n a c~ tL y UJ '~ N Z y u1 >- ~j >- W a, a o °
E '-° Q ° H arc m m Q m t- o~ ~'.' d m m m ~ f- m r- z O ~-
m J' O Z J ~ t
~uf ? Z ~ Z Z tll Z_ N a Z_ r' Z ~ Z_ 111 ~ t0 d ~ X07 Z ~ J Z 111 Z_ I-.- ~ ~
Z ~ ~ O C~ a CO O [L o
d ~ v~'.r=~~~YmhCLYfLYrsQOmt m ai~CCt~u~Q~DO~~'tti~=mtim~U' vl-
C ° cnZ~~IZ~=~NZ~S~ZZOC~ is U UaLLL-aZa~~~~az~c~a~?"'~ a ~'u
41 X ~ J J ~ -Iw J ~ ~ J Ua.J J W Y F- ~ b ~ N LU JIz Ill YIllJ ~h- =IIIJ Y/LL
IL ml~Ill.
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m N h h 'Q' (p ~ M O C ~ Q ~ (O h h ~ O ~ 1f7 f7 Ln W C'~7 N I N ~ O
h h h m N fD 11 (D tn N O O O~tp M O O UM5 C~I~ h N~C'~~ V ~' N
d111N~MpOmNtOO~ OIlIIILMO~~ O
Q ~~Q ~ 7 Q
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1 I
,I
CA 02290738 1999-11-19
WO 98153103 PCT/US98/10561
TABLE S (CONT)
-130-
CA 02290738 1999-11-19
WO 98/53103 PCT/US98/105b1
Ce!! Cycle Array
In the cell cycle array according to the subject invention, all of the unique
polynucleotide probe compositions correspond to genes that are associated with
the life cycle
of a cell. In a specific cell cycle array of interest, the spots are as
provided in Table 9.
-131-
CA 02290738 1999-11-19
WO 98/53103 PCT/US98/10561
TABLE 9
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-132-
CA 02290738 1999-11-19
WO 98/53103 PCT/US98/10561
TABLE 9 (CONT)
I Ii I I
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-133-
CA 02290738 1999-11-19
WO 98/53103 PCT/US98/10561
TA8LE 9 (CONT)
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-134-
CA 02290738 1999-11-19
WO 98/53103 PCT/US98/10561
TABLE 9 (CONT)
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-t3~-
CA 02290738 1999-11-19
WO 98/53103 PCT/US98/10561
()then Reprc~,sentcrtive Arruys
In a neuroarray according to the subject invention, all of the unique
polynucleotide
probe compositions will correspond to genes that are expressed in brain
related tissues.
Genes that are represented on the array are key genes, by which is meant that
they have been
reported to play primary roles in a variety of different biological processes
in brain tissues.
Genes of interest that may be represented on the array include: ion
channel/transport
proteins; receptors; cell cycle regulators; stress response proteins;
apoptosis proteins; signal
transduction proteins; transcriptional factors; growth
factors/interleukins/hormones;
oncogenes and tumor suppressors; cell surface/adhesion proteins; DNA
I O synthesis/repair/recombination genes; and metabolic pathway enzymes.
In certain embodiments, of particular interest is an array having the
following types
of genes represented on its surface: nuclear proteins; endoplasmic reticulum
proteins; golgi
complex proteins; endosomal proteins; lysosomal proteins; peroxisomal
proteins;
mitochondrial proteins; cytoplasmic proteins; cytoskeletal proteins; plasma
membrane
I 5 proteins; post synaptic and dendritic proteins; axonal and nerve terminal
proteins; secreted
proteins, neuropeptides, hormones and growth factors; extracellular matrix
proteins;
astrocyte and oligodendroglial proteins; immune system proteins;
developmentally regulated
proteins; regionally regulated proteins; and disease related proteins.
Other representative arrays include: ( 1 ) rat arrays, in which each of the
unique
20 polynucleotide corresponds to a key rat gene; (?) blood arrays, in which
each unique
polynucleotide corresponds to a gene associated cells and tissues associated
with the
cardiovascular system; (3) rat stress arrays; and (4) mouse stress arrays, in
which each
unique polynucleotide corresponds to a gene associated with the stress
response of murine
cells.
METHODS OF USING THE SUBJECT ARRAYS
The subject arrays find use in a variety of different applications in which
one is
interested in detecting the occurrence of one or more binding events between
target nucleic
acids and probes on the array and then relating the occurrence of the binding
events) to the
presence of a targets) in a sample. In general, the device will be contacted
with the sample
suspected of containing the target under conditions sufficient for binding of
any target
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present in the sample to a complementary polynucleotide present on the array.
Generally, the
sample will be a fluid sample and contact will be achieved by introduction of
an appropriate
volume of the fluid sample onto the array surface, where introduction can be
pipette,
deposition, and the like.
Generation ojLabeled Target
Targets may be generated by methods known in the art. mRNA can be labeled and
used directly as a target, or converted to a labeled cDNA target. Generally,
such methods
include the use of oligonucleotide primers. Primers that may be employed
include oligo dT,
random primers, e.g. random hexamers and gene specific primers.
Of particular interest in the generation of labeled target is the use of a set
of a
representational number of gene specific primers, as described in U.S. Patent
Application
No. 08/ 859,998, the disclosure of which is herein incorporated by reference.
As the subject
sets comprise a representational number of primers, the total number of
different primers in
any given set will be only a fraction of the total number of different or
distinct RNAs in the
sample, where the total number of primers in the set will generally not exceed
80 %, usually
will not exceed 50 % and more usually will not 20% of the total number of
distinct RNAs,
usually the total number of distinct messenger RNAs (mRNAs), in the sample.
Any two
given RNAs in a sample will be considered distinct or different if they
comprise a stretch of
at least 100 nucleotides in length in which the sequence similarity is less
than 98%, as
measured using the FASTA algorithm at default settings. As the sets of gene
specific primers
comprise only a representational number of primers, with physiological sources
comprising
from 5,000 to 50,000 distinct RNAs, the number of different gene specific
primers in the set
of gene specific primers will typically range from about 20 to 10,000, usually
from 50 to
2,000 and more usually from 75 to 1500.
Each of the gene specific primers of the sets described above will be of
sufficient
length to specifically hybridize to a distinct nucleic acid member of the
sample, e.g. RNA or
c DNA, where the length of the gene specific primers will usually be at least
8 nt, more
usually at feast 20 nt and may be as long as 25 nt or longer, but will usually
not exceed 50 nt.
The gene specit7c primers will be sufficiently specific to hybridize to
complementary
template sequence during the generation of labeled nucleic acids under
conditions sufficient
for first strand cDNA synthesis, which conditions are known by those of skill
in the art. The
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number of mismatches between the gene specific primer sequences and their
complementary
template sequences to which they hybridize during the generation of labeled
nucleic acids in
the subject methods will generally not exceed 20 number %, usually will not
exceed 10
number % and more usually will not exceed 5 number %.
Generally, the sets of gene specific primers will comprise primers that
correspond to
at least 20, usually at least 50 and more usually at least 75 distinct genes
as represented by
distinct mRNAs in the sample, where the term "distinct" when used to describe
genes is as
defined above, where any two genes are considered distinct if they comprise a
stretch of at
least 100 nt in their RNA coding regions in which the sequence similarity does
not exceed
98%, as determined using the FASTA algorithm at default settings.
The gene specific oligonucleotide primers may be synthesized by conventional
oligonucleotide chemistry methods, where the nucleotide units may be: (a)
solely nucleotides
comprising the heterocyclic nitrogenous bases found in naturally occurring DNA
and RNA,
e.g. adenine, cytosine, guanine, thymine and uracil; (b) solely nucleotide
analogs which are
capable of base pairing under hybridization conditions in the course of DNA
synthesis such
that they function as the above nucleotides found in naturally occurring DNA
and RNA,
where illustrative nucleotide analogs include inosine, xanthine, hypoxanthine,
1,2-
diaminopurine and the like; or (c) from combinations of the nucleotides of (a)
and nucleotide
analogs of (b), where with primers comprising a combination of nucleotides and
analogues
thereof, the number of nucleotide analogues in the primers will typically be
less than 25 and
more typically less than 5. The gene specific primers may comprise reporter or
hapten
groups, usually 1 to 2, which serve to improve hybridization properties and
simplify
detection procedure.
Depending on the particular point at which the gene specific primers are
employed in
the generation of the labeled nucleic acids, e.g. during first strand cDNA
synthesis or
following one or more distinct amplification steps, each gene specific primer
may
correspond to a particular RNA by being complementary or similar, where
similar usually
means identical, to the particular RNA. For example, where the gene specific
primers are
employed in the synthesis of first strand cDNA, the gene specific primers will
be
complementary to regions of the RNAs to which they correspond.
Each gene specific primer can be complementary to a sequence of nucleotides
which
is unique in the population of nucleic acids, e.k. mRNAs, with which the
primers are
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contacted, or one or more of the gene specific primers in the set may be
complementary to
several nucleic acids in a given population, e.g. multiple mRNAs, such that
the gene specific
primer generates labeled nucleic acid when one or more of set of related
nucleic acid species,
e.g. species having a conserved region to which the primer corresponds, are
present in the
sample. Examples of such related nucleic acid species include those
comprising: repetitive
sequences, such as Alu repeats, A1 repeats and the like; homologous sequences
in related
members of a gene-family; polyadenylation signals; splicing signals; or
arbitrary but
conversed sequences.
Depending on the particular nature of the labeled nucleic acid generation step
of the
subject methods, the gene specific primers may be modified in a variety of
ways. One way
the gene specific primers may be modified is to include an anchor sequence of
nucleotides,
where the anchor is usually located 5' of the gene specific portion of the
primer and ranges in
length from 10 to 50 nt in length, usually 15 to 40 nt in length. The anchor
sequence may
comprise a sequence of bases which serves a variety of functions, such as a
sequence of
bases which correspond to the sequence found in promoters for bacteriophage
RNA
polymerase, e.g. T7 polymerase, T3 poiymerase, SP6 polymerase, and the,like;
arbitrary
sequences which can serve as subsequent primer binding sites; and the like.
Turning now to the methods employing the above sets of gene specific primers,
the
first step in the subject methods is to obtain a sample of nucleic acids,
usually RNAs, from a
physiological source, usually a plurality of physiological sources, where the
term plurality is
used to refer to 2 or more distinct physiological sources. The physiological
source of RNAs
will typically be eukaryotic, with physiological sources of interest including
sources derived
single celled organisms such as yeast and multicellular organisms, including
plants and
animals, particularly mammals, where the physiological sources from
multicellular
2~ organisms may be derived from particular organs or tissues of the
multicellular organism, or
from isolated cells derived therefrom. Thus, the physiological sources may be
different cells
from different organisms of the same species, e.g. cells derived from
different humans, or
cells derived from the same human (or identical twins) such that the cells
share a common
genome, where such cells will usually be from different tissue types,
including normal and
diseased tissue types, e.g. neoplastic, cell types. In obtaining the sample of
RNAs to be
analyzed from the physiological source from which it is derived, the
physiological source
may be subjected to a number of different processing steps, where such
processing steps
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might include tissue homogenation, nucleic acid extraction and the like, where
such
processing steps are known to the those of skill in the art. Methods of
isolating RNA from
cells, tissues, organs or whole organisms are known to those of skill in the
art and are
described in Maniatis et al., Molecular Cloning: A Laboratory Manual (Cold
Spring Harbor
Press)( 1989).
The next step in the subject methods is the generation of labeled nucleic
acids
representative of the nucleic acid, usually RNA, profile of the physiological
source. As
mentioned above, a set of gene specific primers is used to generate the
labeled nucleic acids
from the sample of RNAs, where the labeled nucleic acids generated in this
step may serve
as "target" in subsequent assays in which the differences in the RNA profiles
of at least two
sources are analyzed. As used herein, the term "target" refers to single
stranded RNA, single
stranded DNA and double stranded DNA, where the target is generally greater
than 50 nt in
length.
The set of primers may be used either in first strand cDNA synthesis or
following
one or more amplification steps. Furthermore, the actual synthesis of the
labeled nucleic
acids may be at the same step during which the sets of gene specific primers
are employed,
or the synthesis of the labeled nucleic acids may be one more steps subsequent
to the step in
which the sets of gene specific primers are employed.
In a first embodiment of the invention, the set of gene specific primers is
used to
generate labeled first strand cDNA, where the labeled first strand cDNA is
representative of
the RNA profile of the physiological source being assayed. The labeled first
strand cDNA is
prepared by contacting the RNA sample with the primer set and requisite
reagents under
conditions sufficient for reverse transcription of the RNA template in the
sample. Requisite
reagents contacted with the primers and RNAs are known to those of skill in
the art and will
generally include at least an enzyme having reverse transcriptase activity and
dNTPs in an
appropriate buffer medium.
A variety of enzymes, usually DNA polymerases, possessing reverse transcnptase
activity can be used for the first strand cDNA synthesis step. Examples of
suitable DNA
polymerases include the DNA polymerases derived from organisms selected from
the group
consisting of a thermophilic bacteria and archaebacteria, retroviruses,
yeasts, Neurosporas,
Drosophilas, primates and rodents. Preferably, the DNA polymerase will be
selected from
the group consisting of Moloney marine leukemia virus (M-MLV) as described in
United
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WO 98/53103 PCT/US98/10561
States Patent No. 4,943,531 and M-MLV reverse transciptase lacking RNaseH
activity as
described in United States Patent No. 5,405,776 (the disclosures of which
patents are herein
incorporated by reference), human T-cell leukemia virus type I ( HTLV-I ),
bovine leukemia
virus ( BLV ), Rous sarcoma virus (RSV ), human immunodeficiency virus ( HIV )
and
Thermus aquaticus ( Taq ) or Thermus thermophilus (Tth) as described in United
States
Patent No. 5,322,770, the disclosure of which is herein incorporated by
reference. Suitable
DNA polymerises possessing reverse transcriptase activity may be isolated from
an
organism, obtained commercially or obtained from cells which express high
levels of cloned
genes encoding the polymerises by methods known to those of skill in the art,
where the
particular manner of obtaining the polymerise will be chosen based primarily
on factors such
as convenience, cost, availability and the like.
The various dNTPs and buffer medium necessary for first strand cDNA synthesis
through reverse transcription of the primed RNAs may be purchased commercially
from
various sources, where such sources include Clontech, Sigma, Life
Technologies,
Amersham, Boehringer-Mannheim. Buffer mediums suitable for first strand
synthesis will
usually comprise buffering agents, usually in a concentration ranging from 10
to 100 pM
which typically support a pH in the range 6 to 9, such as Tris-HC1, HEPES-KOH,
etc.; salts
containing monovalent ions, such as KC1, NaCI, etc., at concentrations ranging
from 0-200
mM; salts containing divalent cations like MgCh, Mg(OAc) etc, at
concentrations usually
ranging from i to 10 mM; and additional reagents such as reducing agents, e.g.
DDT,
detergents, albumin and the like. The conditions of the reagent mixture will
be selected to
promote efficient first strand synthesis. Typically the set of primers will
first be combined
with the RNA sample at an elevated temperature, usually ranging from 50 to 95
°C,
followed by a reduction in temperature to a range between about 0 to
60°C, to ensure
specific annealing of the primers to their corresponding RNAs in the sample.
Following this
annealing step, the primed RNAs are then combined with dNTPs and reverse
transcriptase
under conditions sufficient to promote reverse transcription and first strand
cDNA synthesis
of the primed RNAs. By using appropriate types of reagents, all of the
reagents can be
combined at once if the activity of the polymerise can be postponed or timed
to start after
annealing of the primer to the RNA.
In this embodiment. one of either the gene specific primers or dNTPs,
preferably the
dNTPs, will be labeled such that the synthesized cDNAs are labeled. By labeled
is meant
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that the entities comprise a member of a signal producing system and are thus
detectable,
either directly or through combined action with one or more additional members
of a signal
producing system. Examples of directly detectable labels include isotopic and
fluorescent
moieties incorporated into, usually covalently bonded to, a nucleotide
monomeric unit, e.g.
dNTP or monomeric unit of the primer. Isotopic moieties or labels of interest
include''-P,
33P, ass, ~zsl, ~d the like. Fluorescent moieties or labels of interest
include coumarin and its
derivatives, e.g. 7-amino-4-methylcoumarin, aminocoumarin, bodipy dyes, such
as Bodipy
FL, cascade blue, fluorescein and its derivatives, e.g. fluorescein
isothiocyanate, Oregon
green, rhodamine dyes, e.g. texas red, tetramethylrhodamine, eosins and
erythrosins, cyanine
dyes, e.g. Cy3 and CyS, macrocyclic chelates of lanthanide ions, e.g. quantum
dyer"',
fluorescent energy transfer dyes, such as thiazole orange-ethidium
heterodimer, TOTAB, etc.
Labels may also be members of a signal producing system that act in concert
with one or
more additional members of the same system to provide a detectable signal.
Illustrative of
such labels are members of a specific binding pair, such as ligands, e.g.
biotin, fluorescein,
digoxigenin, antigen, polyvalent cations, chelator groups and the like, where
the members
specifically bind to additional members of the signal producing system, where
the additional
members provide a detectable signal either directly or indirectly, e.g.
antibody conjugated to
a fluorescent moiety or an enzymatic moiety capable of converting a substrate
to a
chromogenic product, e.g. alkaline phosphatase conjugate antibody; and the
like.
In one preferred embodiment, the member of the signal producing system bound
to
the nucleotide is functional group capable of covalently binding to additional
members of the
signal producing system to generate a detectable label. Examples of such
functional groups
or moieries include amino, sulfhydryl, azido, isothiocyanate, sulfoxyl, and
the like. The
labeled target generated using such nucleotides will thus include one or more,
usually a
plurality of, functional moieties. For detection, the functional moieties of
the modified
nucleotides can be labeled by conjugation of a label to the functional moiety.
A variety of
suitable labels and methods for their conjugation to functional moieties are
known to those
of skill in the art. Examples include labeling of amino-modified cDNA by a
succinimidyl
ester of an appropriate dye, e.g. Alexa, Bodipy, or Cy dyes. Alternatively,
label can be
entrapped or bonded into structures of microscopic-sized particles. These
particles can then
be conjugated with the functional moieties of the target.
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WO 98/53103 PCT/US98/10561
For each sample of RNA, one can generate labeled oligos with the same labels.
Alternatively, one can use different labels for each physiological source,
which provides for
additional assay configuration possibilities, as described in greater detail
below.
In a variation of the above embodiment, where desired one can generate labeled
RNA
instead of labeled first strand cDNA. In this embodiment. first strand cDNA
synthesis is
carried out in the presence of unlabeled dNTPs and unlabeled gene specific
primers.
However, the primers are optionally modified to comprise a promotor for an RNA
polymerise, such as T7 RNA polymerise, T3 RNA polymerise, SP6 RNA polymerise,
and
the like. In this embodiment, following first strand cDNA synthesis, the
resultant single
stranded cDNA is then converted to double stranded cDNA, where the resultant
double
stranded cDNA comprises the anchor sequence comprising the promoter region.
Conversion
of the mRNA:cDNA hybrid following first strand synthesis can be carried out as
described
in Okayama & Berg, Mol. Cell. Biol. (1982) 2:161-170, and Gubler & Hoffman,
Gene
(1983) 25: 253-269, where briefly the RNA is digested with a ribonuclease,
such as E.coli
RNase H, followed by repair synthesis using a DNA polymerise like DNA
polymerise I,
etc., and E.coli DNA ligase. One may also employ the modification of this
basic method
described in Wu, R, ed., Methods in Enzymology ( 1987), vol. 153 (Academic
Press). Next,
the double stranded cDNA is contacted with RNA polymerise and dNTPs, including
labeled
dNTPs as described above, to produce linearly amplified labeled ribonucleic
acids. For
cDNA lacking the anchor sequence comprising a promoter region, a polymerise
that does
not need a promoter region but instead can initiate RNA strand synthesis
randomly from
cDNA, such as core fragment of E.Coli RNA polymerise, may be employed.
In another embodiment of the subject invention, the labeled nucleic acid
generation
step comprises one or more enzymatic amplification steps in which multiple DNA
copies of
the initial RNAs present in the sample are produced, from which multiple
copies of the
initial RNA or multiple copies of antisense RNA (aRNA) may be produced, using
the
polymerise chain reaction, as described in U.S. Pat. No. 4,683,195, the
disclosure of which
is herein incorporated by reference, in which repeated cycles of double
stranded DNA
denaturation, oligonucieotide primer annealing and DNA polymerise primer
extension are
performed, where the PCR conditions may be modified as described in U.S. Pat
No.
5,436,149, the disclosure of which is herein incorporated by reference.
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In one embodiment involving enzymatic amplification, the set of gene-specific
primers are employed in the generation of the first strand cDNA, followed by
amplification
of the first strand cDNA to produce amplified numbers of labeled cDNA. In this
embodiment, as a set of gene-specific primers is employed in the first strand
synthesis step,
only a representative proportion of the total RNA in the sample is amplified
during the
subsequent amplification steps.
Amplification of the first strand cDNA can be conveniently achieved by using a
CAPswitchT"' oligonucleotide as described in U.S. Patent Application Serial
No. 08!582,562,
the disclosure of which is herein incorporated by reference. Briefly, the
CAPswitchTM
technology uses a unique CAPswitchT"' oligonucleotide in the first strand cDNA
synthesis
followed by PCR amplification in the second step to generate a high yield of
ds cDNA.
When included in the first-strand cDNA synthesis reaction mixture, the
CAPswitchT"~
oligonucleotide serves as a short extended template. When reverse
transcriptase stops at the
5' end of the mRNA template in the course of first strand cDNA synthesis it
switches
templates and continues DNA synthesis to the end of the CAPswitchTM
oligonucleotide. The
resulting ss cDNA incorporates at the 3' end, sequence which is complimentary
to complete
5' end of the mRNA and the CAPswitchTM oligonucleotide sequence.
Of particular interest as the CAPswitchTM oligonucleotide are oligonucleotides
having the following formula:
5'-dN 1-dN2-...dNm-rN 1-rN2...rNn-3'
wherein:
dN represents a deoxyribonucleotide selected from among dAMP, dCMP, dGMP and
dTMP;
m represents an integer 0 and above, preferably from l0 to 50;
rN represents a ribonucleotide selected from the group consisting of AMP, CMP,
GMP and UMP, preferably GMP; and
n represents an integer 0 and above, preferably from 3 to 7.
The structure of the CAPswitchTM oligonucleotide may be modified in a number
of
ways, such as by replacement of 1 to l0 nucleotides with nucleotide analogs,
incorporation
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of terminator nucleotides, such as 3'-amino NMP, 3'-phosphate NMP and the
like, or
non-natural nucleotides which can improve efficiency of the template switching
reaction but
still retain the main function of the CAPswitchTM oligonucleotide i.e. CAP-
depended
extension of full-length cDNA by reverse transcriptase using CAPswitchT"'
oligonucleotide
as a template.
In using the CAPswitchTM oligonucleotide, first strand cDNA synthesis is
carried out
in the presence of a set of gene specific primers and a CAPswitchT"''
oligonucleotide, where
the gene specific primers have been modified to comprise an arbitrary anchor
sequence at
their 5' ends. The first strand cDNA is then combined with primer sequences
complementary
to: (a) all or a portion of the CAPswitchTM oligonucleotide and (b) the
arbitrary anchor
sequence of the gene specific primers and additional PCR reagents, such as
dNTPs, DNA
polymerase, and the like, under conditions sufficient to amplify the first
strand cDNA.
Conveniently, PCR is carried out in the presence of labeled dNTPs such that
the resultant,
amplified cDNA is labeled and serves as the labeled or target nucleic acid.
Labeled nucleic
acid can also be produced by carrying out PCR in the presence of labeled
primers, where
either or both the CAPswitchTM oligonucleotide complementary primer and anchor
sequence
complementary primer may be labeled. In yet an alternative embodiment, instead
of
producing labeled amplified cDNA, one may generate labeled RNA from the
amplified ds
cDNA, e.g. by using an RNA polymerase such as E.coli RNA polymerase, or other
RNA
polymerases requiring promoter sequences, where such sequences may be
incorporated into
the arbitrary anchor sequence.
Instead of using the set of gene specific primers in the first strand cDNA
synthesis
step followed by subsequent amplification of only a representative fraction of
the total
number of distinct RNA species in the sample, one may also amplify all of the
RNAs in the
sample and use the set of gene specific primers to generate labeled nucleic
acid following
amplification. This embodiment may find use in situations where the RNA of
interest to be
amplified is known or postulated to be in small amounts in the sample.
In this embodiment, first strand synthesis is carried out using: (a) an oligo
dT primer
that usually comprises an arbitrary anchor sequence at its 5' end and (b) a
CAPswitchTM
oligonucleotide. During first strand synthesis the oligo(dT) anneals to the
polyA tail of the
mRNA in the sample and synthesis extends beyond the 3' end of the RNA to
include the
CAPswitchT"' oligonucleotide, yielding a first strand cDNA comprising an
arbitrary
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sequence at its 5' end and a region complementary to the CAPswitchT"'
oligonucleotide at its
3' end. The length of the dT primer will typically range from 15 to 30 nts,
while the arbitrary
anchor sequence or portion of the primer will typically range from 15 to 25 nt
in length.
Following first strand synthesis, the cDNA is amplified by combining the first
strand
eDNA with primers that correspond at least partially to the anchor sequence
and the
CAPswitchT"' oligonucleotide under conditions sufficient to produce an
amplified amount of
the cDNA. Labeled nucleic acid is then produced by contacting the resultant
amplified
cDNA with a set of gene specific primers, a polymerise and dNTPs, where at
least one of the
gene specific primers and dNTPs are labeled.
When employed to generate target, as described above, the gene specific
primers of
the sets of primers according to the subject invention are typically chosen
according to a
number of different criteria. In some embodiments of the invention, primers of
interest for
inclusion in the set include primers corresponding to genes which ire
typically differentially
expressed in different cell types, in disease states, in response to the
influence of external
agents, factors or infectious agents, and the like. In other embodiments,
primers of interest
are primers corresponding to genes which are expected to be, or already
identified as being,
differentially expressed in different cell, tissue or organism types.
Preferably, at least 2
different gene functional classes will be represented in the sets of gene
specific primers,
where the number of different functional classes of genes represented in the
primer sets will
generally be at least 3, and will usually be at least 5. Gene functional
classes of interest
include oncogenes; genes encoding tumor suppressors; genes encoding cell cycle
regulators;
stress response genes; genes encoding ion channel proteins; genes encoding
transport
proteins; genes encoding intracellular signal transduction modulator and
effector factors;
apoptosis related genes; DNA synthesis/recombination/repair genes; genes
encoding
transcription factors; genes encoding DNA-binding proteins; genes encoding
receptors,
including receptors for growth factors, chemokines, interleukins, interferons,
hormones,
neurotransmitters, cell surface antigens, cell adhesion molecules etc.; genes
encoding cell-
cell communication proteins, such as growth factors, cytokines, chemokines,
interleukins,
interferons, hormones etc.; and the like. Less preferred are gene specific
primers that are
subject to formation of strong secondary structures with less than -I
Okcal/mol; comprise
stretches of homopolymeric regions, usually more than 5 identical nucleotides;
comprise
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more than 3 repetitive sequences; have high, c'. s,~. more than 80%, or low,
c'. ~~. less than 30%,
GC content etc.
The particular genes represented in the set of gene specific primers will
necessarily
depend on the nature of physiological source from which the RNAs to be
analyzed are
derived. For analysis of RNA profiles of eukaryotic physiological sources, the
genes to
which the gene specific primers correspond will usually be Class II genes
which are
transcribed into RNAs having S' caps, e.g. 7-methyl guanosine or 2,2,7-
trimethylguanosine,
where Class II genes of particular interest are those transcribed into
cytoplasmic mRNA
comprising a 7-methyl guanosine 5' cap and a polyA tail.
For analysis of RNA profiles of mammalian physiological sources, of particular
interest are gene specific primers corresponding to the functional gene
classes listed above.
For analysis of RNA profiles of human physiological sources, the gene specific
primers of
particular interest are the gene specific primers identified in Table 1 as SEQ
ID NO:O1 to
SEQ ID NO:I372, of U.S. Application Serial No. 08/859,998, the disclosure of
which is
herein incorporated by reference, where sets of these primers will usually
include at least 20
and more usually at least SO of these specific sequences.
Particular sets of primers of interest in the subject invention are those sets
of primers
that include primers capable of amplifying at least a portion of the unique
polynucleotides
present on the arrays with which the target is to be employed. By at least a
portion is meant
at least about 10, usually at least about 20 and more usually at least about
25 number
(where number is the number of different unique polynucleotides on the array).
For
examples, sets of primers that include primers capable of amplifying at least
a portion of the
unique polynucleotides listed in Table 1, supra, are of interest. Similarly
sets of primers
capable of amplifying at least a portion of the unique polynucleotides listed
in Tables 2 to 8,
.supra, are also of interest.
In a particularly preferred embodiment, the gene specific primers are
preferably those
primers that correspond to the different polynucleotide spots on the array
that is used in the
hybridization assay. Thus, one will preferably employ gene specific primers
for each
different polynucleotide that is present on the array, so that if the gene is
expressed in the
particular cell or tissue being analyzed, labeled target will be generated
From the sample for
that gene. In many embodiments in which the subject arrays are employed, the
gene specific
primers used to generate the target from the human cell or tissue being
analyzed will have
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the same sequence as the gene specific primers used to generate the
poiynucleotide probes
present on the array. In this manner, if a particular gene present on the
array is expressed in a
particular sample, the appropriate target will be generated and subsequently
identified.
Representative sets of primers falling within this particularly preferred
embodiment include:
SET DESCRIPT10N
I pair of primers capable of amplifying each
polynucleotide listed in Table I, supra, as well
one set of primers capable of amplifying each of
the complementary sequences of each of the
polynucieotides listed in Table 1.
1 pair of primers capable of amplifying each
polynucleotide listed in Table 2, supra, as well
one set of primers capable of amplifying each of
the complementary sequences of each of the
polynucleotides listed in Table 2.
I pair of primers capable of amplifying each
polynucleotide listed in Table 3, supra, as well
one set of primers capable of amplifying each of
the complementary sequences of each of the
polynucleotides listed in Table 3.
Hybridization and Detection
As mentioned above, following preparation of the target nucleic acid from the
tissue
or cell of interest, the target nucleic acid is then contacted with the array
under hybridization
conditions, where such conditions can be adjusted, as desired, to provide for
an optimum
level of specificity in view of the particular assay being performed. Suitable
hybridization
conditions are well known to those of skill in the art and reviewed in
Maniatis et al, supra
and WO 95/21944. In analyzing the differences in the population of labeled
target nucleic
acids generated from two or more physiological sources using the arrays
described above,
each population of labeled target nucleic acids are separately contacted to
identical probe
arrays or together to the same array under conditions of hybridization,
preferably under
stringent hybridization conditions (for example, at SO°C or higher and
O.1XSSC (15 mM
sodium chloride/O1.5 mM sodium citrate)) , such that labeled target nucleic
acids hybridize
to complementary probes on the substrate surface.
Where all of the target sequences comprise the same label, different arrays
will be
employed for each physiological source (where different could include using
the same array
at different times). Alternatively, where the labels of the targets are
different and
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distinguishable for each of the different physiological sources being assayed,
the opportunity
arises to use the same array at the same time for each of the different target
populations.
Examples of distinguishable labels are well known in the art and include: two
or more
different emission wavelength fluorescent dyes, like Cy3 and CyS, two or more
isotopes
with different energy of emission, like''-P and "P, labels which generate
signals under
different treatment conditions, like temperature, pH, treatment by additional
chemical agents,
etc., or generate signals at different time points after treatment. Using one
or more enzymes
for signal generation allows for the use of an even greater variety of
distinguishable labels,
based on different substrate specificity of enzymes (alkaline
phosphatase/peroxidase).
Following hybridization, non-hybridized labeled nucleic acid is removed from
the
support surface, conveniently by washing, generating a pattern of hybridized
nucleic acid on
the substrate surface. A variety of wash solutions are known to those of skill
in the art and
may be used.
The resultant hybridization patterns of labeled nucleic acids may be
visualized or
detected in a variety of ways, with the particular manner of detection being
chosen based on
the particular label of the target nucleic acid, where representative
detection means include
scintillation counting, autoradiography, fluorescence measurement,
colorimetric
measurement, light emission measurement and the like.
Following detection or visualization, the hybridization patterns may be
compared to
identify differences between the patterns. Where arrays in which each of the
different probes
corresponds to a known gene are employed, any discrepancies can be related to
a differential
expression of a particular gene in the physiological sources being compared.
Utility
The subject methods find use in, among other applications, differential gene
expression assays. Thus, one may use the subject methods in the differential
expression
analysis of: (a} diseased and normal tissue, e.g. neoplastic and normal
tissue, (b) different
tissue or tissue types; (c) developmental stage; (d) response to external or
internal stimulus;
(e) response to treatment; and the like. The subject arrays therefore find use
in broad scale
expression screening for drug discovery and research, such as the effect of a
particular active
agent on the expression pattern of genes in a particular cell, where such
information can be
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used to reveal drug toxicity, carcinogenicity, etc., environmental monitoring,
disease
research and the like.
KiTs
Also provided are kits for performing anaiyte binding assays using the subject
devices, where kits for carrying out differential gene expression analysis
assays are
preferred. Such kits according to the subject invention will at least comprise
the subject
arrays. The kits may further comprise one or more additional reagents employed
in the
various methods, such as primers for generating target nucleic acids,
including a set of gene
l 0 specific primers according to the subject invention, e.g. primer sets 1 to
9 described above,
dNTPs and/or rNTPs, which may be either premixed or separate, one or more
uniquely
labeled dNTPs and/or rNTPs, such as biotinylated or Cy3 or Cy5 tagged dNTPs,
or other
post synthesis labeling reagent, such as chemically active derivatives of
fluorescent dyes,
enzymes, such as reverse transcriptases, DNA polymerases, and the like,
various buffer
mediums, e.g. hybridization and washing buffers, prefabricated probe arrays,
labeled probe
purification reagents and components, like spin columns, etc., signal
generation and
detection reagents, e.g. streptavidin-alkaline phosphatase conjugate,
chemifluorescent or
chemiluminescent substrate, and the like.
The following examples are offered by way of illustration and not by way of
limitation.
EXPERIMENTAL
Example 1 - Generation of human cDNA array
686 cDNA fragments corresponding 686 different human genes were amplified from
quick-clone cDNA (CLONTECH) in 686 separate test tubes using a combination of
sense
and antisense gene-specific primers: (Set No. 9, described supra).
AmpliFcation was
conducted in a 100-pl volume containing 2 pl of mixture of 10 Quick-clone cDNA
from
placenta, brain, liver, lung, leukocytes, spleen, skeletal muscle, testis,
kidney and ovary
(CLONTECH), 40 mM Tricine-KOI-I (pH 9.2 at 22°C), 3.5 mM Mg(OAc),, 10
mM KOAc,
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75 ~tg/ml BSA, 200 ~tM of each dATP, dGTP, dCTP and dTTP, 0.2 ~tM of each
sense and
antisense gene-specific primers and 2 ~tl of KIenTaq Polymerase mix.
Temperature
parameters of the PCR reactions were as follows: 1 min at 95°C followed
by 20-35 cycles of
95°C for 15 sec and 68°C for 2 min; followed by a 10-min final
extension at 68°C. PCR
products were examined on 1.2% agarose/EtBr gels in 1 x TBE buffer. As a DNA
size
marker a 1 Kb DNA Ladder was used. ds cDNA was then precipitated by addition
of a half
volume of 4M ammonium acetate (about 35 pl) and 3.7 volumes of 95% ethanol
(about 260
~tl). After vortexing, the tube was immediately centrifuged at 14,000 r.p.m.
in a
microcentrifuge for 20 min. The pellet was washed with 80% ethanol without
vortexing,
centrifuged as above for 10 min, air dried, and dissolved in 10 ~tl of
deionized water. Yield
of ds cDNA after the amplification step was about 5 ~tg. The ds cDNA fragments
for all 686
genes were cloned into TA-cloning vector using the manufacturer's
recommendations
(Invitrogen) and identity of the clones was confirmed by sequence analysis.
The ds cDNA
inserts with the sequence corresponding 686 genes were amplified by PCR using
a
combination of antisense and sense gene-specific primers, as described above.
The ds cDNA
was denatured by adding 1 ~tl of IOX denaturing solution (I M NaOH, 10 mM
EDTA) and
incubating at 65 °C for 20 min. All cDNA probes were transferred in 384-
well plate and
loaded on positively charged nylon membrane (Schleher & Schull) using 384 pin
tool and
Biomek 2000 (Beckman) robot. The resultant array is described in Table 1.
Example 2 - Generation of''-P-labeled oli$onucleotides during first strand
cDNA
s~ thesis
Step A. cDNA synthesis/Labeling Procedure
I ~tg of polyA+RNA or total RNA was converted into ''-P-labeled first-strand
cDNA
as follows. A sufficient volume of master mix for all labeling reactions and 1
extra reaction
was prepared as follows to ensure sufficient volume. For each 10-~tl labeling
reaction, the
following reagents were mixed:
2 pl SX First-strand buffer (250 pM Tris-HCI pH8.3; 375 mM KCI; IS mM M~CI=)
1 pl I OXdNTP mix (~00 pM dGTP, 500 IrM dCTP, 500 pM dTTP, 5 pM dATP)
4 pl [a-'=P]dATP (Amersham, 3000 Ci/mmol, 10 mCi/ml)
I PI MMLV reverse transcriptase (Amersham, 200 units/pl)
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8 pl Final volume
Next, the following reagents were combined in a 0.5-ml PCR test tube:
1 pg ( I-2 pl) polyA+RNA sample
I pl IOx gene-specific primers mix ( 0.2 pM of each oligonucleotide ID No.
2,4,6,8,10,12,.... 1372 from Table l of U.S. Pateni Application Serial No.
08/859,998, the discosure of which is herein incorporated by reference.)
As a control, in separate test tube were mixed 1 ~tg of polyA+RNA sample with
1 ~tl of oligo
dT primer (CDS 1, 5'-d(TCTAGAATTCAGCGGCCGC(T)3oVN) - 3'
(where V=G or A or C; N=G or A or T or C)
For each tube, ddH~O was added to a final volume of 3 gel and the contents
were
mixed and spun briefly in a microcentrifuge. The tubes were then incubated in
a preheated
PCR thermocycler at 70°C for 2 min. The temperature in thermocycle was
reduced down to
50°C and the tube contents were incubated for 2 min. 8 ul of master mix
as prepared above
were added to each reaction test tube. The contents of the test tubes were
then mixed by
gentle pipetting. The tubes were then incubated in a PCR thermocycler for 20
min at 50°C.
The reaction was then stopped by adding 1 ~tl of l OX termination mix (0.1 M
EDTA, 1
mg/ml glycogen).
Step B. Column Chromatography
The''-P-labeled cDNAs were separated from unincorporated''-P-labeled
nucleotides
and small (<0.1- kb) cDNA fragments using the following procedure for each
test tube. A
CHROMA SPIN-200 column (CLONTECH, Palo Alto, CA) was placed into a 1.5-ml
microcentrifuge tube, the water was allowed to drain through the column by
gravity flow
unfit the surface of the gel beads emerged in the column matrix. The sample
was then
applied to the center of the gel bed's flat surface and allowed to be fully
absorbed into the
resin bed. 25 ~tl of ddH,O were then applied and allowed to completely drain
out of the
column. 200 ~tl of ddH,O were then applied and allowed to completely drain out
of the
column until there was no liquid left above the resin bed. The column was then
transferred to
a clean 1.5-ml microcentrifuge tube.
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To collect the first fraction, 100 ~tl of ddH,O were added to the column and
allowed
to completely drain out of the column. The second, third and fourth fractions
were collected
in analogous fashion. The tubes with fractions 1-4 were then placed in
scintillation counter
empty vials, and Cherenkov counts for each fraction were obtained in the
tritium channel.
The fractions which showed the highest Cerenkov counts were pooled.
Example 3 - Generation of CX3-labeled hybridization ~olvnucleotide target from
polyA+RNA usin~"posts~nthesis labellin~procedure
In this procedure for generating labeled cDNA target, polyA+RNA is first
converted
into cDNA that has primary amino groups which are subsequently coupled with
Cy3
succinimide ester. This technology allows for a significant increase (about 10
fold) in
activity of labeled polynucleotide target and therefore increases the overall
sensitivity of
detection of gene expression. The same procedure can be used for labeling two
(or more)
samples of RNA. In this case the cDNA synthesis step was the same for both
samples but at
the labeling step, each cDNA sample was labeled by different and
distinguishable labels, e.g.
Cy3 and CyS, Alexa 532 and Bodipy TR, Fluorescein and tetramethyl rhodamine,
etc. Each
labeled probe was purified separately by column chromatography and, after
normalization,
were combined together in equal ratio and hybridized with a cDNA array. After
hybridization, the detection procedure revealed both dye-labeled hybridized
target
simultaneously, based on the different spectral characteristics (emission
wavelength) of the
fluorescent labels.
A. cDNA synthesis
The 10-~l reaction described below converted 1 ug of polyA+RNA into amino-
modified first-strand cDNA.
For each cDNA synthesis reaction:
Enough master mix for all labeling reactions and 1 extra reaction was prepared
to
ensure sufficient volume.
For each 10-~cl labeling reaction, the following reagents were mixed:
2 ul SX First-strand buffer (250 ~M Tris-HC I pH8.3; 37~ mlvt KC I; ! 5 mM MgC
12)
i ul lOXdNTP mix (500 ~M dGTP, 500 ~M dCTP, 500 ~M dATP, 100 uM dTTP,
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and 100 uM allylamino dUTP )
1 ~I [a-'Z P]dATP (Amersham, 3000 Ci/mmol, 10 mCi/ml)
3 ~I H~O
1 ul MMLV reverse transcriptase (Amersham, 200 units/ul)
8 ul Final volume
2. The following was combined in a 0.5-ml PCR test tube:
I ug ( I-2 ul) polyA+RNA sample
1 ~.l lOx gene-specific primers mix ( 0.2 uM of each oligonucleotide iD No.
2,4,6,8,10,12,....... 1372) (from Table 1 of U.S. Patent Application No.
08/859,998, the disclosure of which is herein incorporated by reference.)
As a control in separate test tube 1 /.cg of poiyA+RNA sample was mixed with 1
ul
1 S of oligo dT primer (SEQ ID NO. 1373 from Table 1 of U.S. Application No.
08/859,998).
3. ddH,O was added to a final volume of 3 ~[.
4. The contents were mixed and the tubes were spun briefly in a
microcentrifuge.
5. The tubes were incubated in preheated PCR thermocycler at 70°C for 2
min.
6. The temperature in the thermocycle was reduced down to 50°C and
incubate for 2
min.
7. 8 ,ul of master mix were added to each reaction test tube.
8. The contents of the test tubes were mixed by gentle pipeting.
9. The tubes were incubated in a PCR thermocycler for 30 min at 50°C.
2~ 10. The reaction was stopped by increasing temperature up to 70°C
for 5 min, then
cooled to 37°C.
11. 1 ul of RNase H (10 units/~.1) was added and the tubes were incubated at
37°C for 15
mm.
12. The reaction was stopped by adding 40 ~1 of termination mix {0.3 M sodium
acetate.
pH 5.0, 1 mMEDTA).
13. An equal volume (50 gel) of phenol/chlorophorm/isoamyl alcohol mix (1: 1:
1/24 v/v)
was added and extraction was performed. Phases were separated by
centrifugation at
14,000 rpm for 10 min.
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14. Upper water phase was collected and cDNA was precipitated by adding 2.5
volumes
(about 120 ~.l) of ethanol.
15. The precipitate was collected by centrifugation at 14,000 rpm for 10 min,
the
supernatant removed and the precipitate was washed with 80% ethanol.
16. The precipitate was air dried and dissolved in 10 ~cl of 0. 1 M sodium
bicarbonate
buffer, pH 9Ø
Step B. Post synthesis labeling procedure.
1. 1 mg of Cy3 succinimide ester was dissolved in 10 ~cf of dimethyl sulfoxide
and 10
,ul of amino-modified cDNA generated at step 16 was added to it.
2. The mixture was incubated at room temperature overnight.
Step C. Column Chromatography
To purify the Cy3-labeled cDNAs from the unconjugated label, the following was
performed for each test tube:
I . CHROMA SPIN-200 column (CLONTECH) was removed from refrigerator and
allowed to warm at room temperature for about 1 hour. The column was inverted
several times to completely resuspend the gel matrix. (Note: Check for air
bubbles in
the column matrix. If bubbles are visible, resuspend the matrix in the in the
column
buffer (ddH,O) by inverting the column again).
2. The bottom cap from the column was removed, and then the top cap was slowly
removed.
3. The column was placed into a 1.5-ml microcentrifuge tube.
4. The water was allowed to drain through the column by gravity flow until the
surfaces
of the gel beads in the column matrix were visible. (The top of the column
matrix
should be at the 0.75-ml mark on the wall of the column. If the column
contains
much less matrix, adjust the volume of the matrix to 0.7~m1 mark using matrix
from
another column.)
5. The collected water was discarded.
6. The sample was applied to the center of the gel bed's flat surface and
allowed to be
fully absorbed into the resin bed. Care was taken not allow any sample to flow
along
the inner wall of the column.
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7. 25 ~I of ddH,O were applied and allowed to completely drain out of the
column.
8. Apply 200 ~l of ddH,O and allow the buffer to completely drain out of the
column
until there was no liquid left above the resin bed.
9. The column was transfered to a clean 1.5-ml microcentrifuge tube.
10. 100 ,ui of ddH,O were added to the column and allowed to completely drain
out of the
column.
1 1. The second, third and fourth fractions were collected by repeating steps
9-10.
12. Cherenkov counts were obtained for each fraction by counting the entire
sample in
the tritium channel.
13. The fractions (usually fractions 2-3) which showed highest Cerenkov counts
were
pooled. Waste column and the fractions (usually fraction 1 and 4) which showed
less
than 10% counts from peak fractions.
Example 4 - HXbridization 3'P-labeled cDNA Tareet with cDNA Arrav
A solution of ExpressHybT"' (CLONTECH) and sheared salmon testes DNA (Sigma)
was prepared by prewarming 15 ml of ExpressHybTM at 50-60°C, heating
1.5 mg of sheared
salmon testes DNA at 95-100°C for 5 min followed by chilling quickly on
ice, and
combining the resultant heat-denatured sheared salmon testes DNA with the
prewarmed
ExpressHybTM
A cDNA Array as produced in Example 1 above was then placed in a hybridization
bottle and 10 ml of the solution prepared above was added to the bottle.
Prehybridization
was performed for 30 min with continuous agitation at 72°C. Labeled
cDNA probe
(Example l, about 200 ul, total about 2-5x106 cpm) with 1/lOth of the total
volume ( about
22 ul) of lOx denaturing solution (1 M NaOH, l0 mM EDTA) was mixed and
incubated at
65°C for 20 min. 5 pl (1 p.g/ul) of human Cot-1 DNA was then added, and
an equal volume
(about 225 ~1) of 2x Neutralizing solution ( 1 M NaHP04, pH 7.0) was added and
incubation
continued at 65°C for 10 min. The mixtures were then combined and
thoroughly mixed.
The prehybridization solution was replaced with the solution comprising the
labeled
oligonucleotide as prepared above and allowed to hybridize overnight with
continuous
agitation at 65°C. Following hybridization, the hybridization solution
was carefully removed
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and discarded, replaced with 200 ml of Wash Solution 1 (2X SSC, 1% SDS). The
array was
washed for 20 min with continuous agitation at 65 °C. Washing was
repeated tour times.
Two additional 20-min washes were then performed in 200 ml of prewarmed Wash
Solution 2 (O.1X SSC, 0.5% SDS) with continuous agitation at 65°C.
Using forceps, the
cDNA array was removed from the container and excess wash solution was removed
by
shaking.
The damp membrane was immediately wrapped in plastic wrap, mounted on
Whatman paper (3mm Chr) and exposed to x-ray film at -70°C with an
intensifying screen.
Example 5 -Comparison~etween Using Sets of Gene Specific Primers and oligo dT
''-P-labeled cDNA target were synthesized by M-MLV reverse transcriptase from
a
mixture 588 antisense gene-specific primers (B) or oiigo dT(A) using placenta
polyA+RNA
as a template as described in Example 2. Primer extension products generated
by reverse
transcription were purified by gel filtration as described in Example 2 and
hybridized
separately with two cDNA arrays comprising 588 human genes under identical
conditions as
described in Example 4. Signals which can be detected by using cDNA target
generated
using the set of gene specific primers but can not be detected by using
conventional target
generated with oligo dT primers were observed. Note, the level of non-specific
background
detected as signal generated by membrane alone outside of the regions with
immobilized
probes generated by target generated using oligo dT primers was significantly
higher in
comparison with the background generated by the target generated by using the
sets of gene
specific primers.
Example 6 - Generation of cDNA array,Drobe immobilized on~lass slides.
SO cDNA fragments corresponding to 50 different human genes were amplified
from
piasmid clones containing corresponding cDNA fragments in 96 well plates using
combination of vector primer ID No. 1376 and ID No. 1377 or sense and
antisense gene-
specific primers: ID No. 1+2, 3+4,5+6,7+8,.... 100+l0I (from Table 1 of U.S.
Patent
Application No. 08/859,998, the disclosure of which is herein incorporated by
reference).
Amplification was conducted in a 400-,ul volume containing 2 ng of plasmid
DNA, 40 mM
Tricine-KOH (pH 9.2 at 22°C), 3.~ mM Mg(OAc),, 10 MM KOAc, 7~ ~g/ml
BSA, 200 ,uM
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of each dATP, dGTP, dCTP and dTTP, 0.2 ~M of each primers and 2 ~l of KIenTaq
Polymerase mix (CLONTECH). Temperature parameters of the PCR reactions were as
follows: 1 min at 95°C followed by 30 cycles of 95°C for 15 sec
and 68°C for 2 min;
followed by a 10-min final extension at 68°C. PCR products were
examined on 1.2%
S agarose/EtBr gels in l x TBE buffer. As a DNA size marker , a 1 Kb DNA
Ladder was used.
ds cDNA was then precipitated by addition of a 10% volume of 3M sodium acetate
(pH 5-0)
(about 40 ~cl) and 2.5 volumes of 96% ethanol (about 1 ml). After vortexing,
the tube was
immediately centrifuged at 14,000 r.p.m. in a microcentrifuge for 20 min. The
pellet was
washed with 80% ethanol without vortexing, centrifuged as above for 10 min,
air dried, and
dissolved in l0,ul of deionized water. Yield of ds cDNA after amplification
step was about
fig. The ds cDNA was solved in I O ,ul of distilled water, 10 ~l of 1 M sodium
carbonate
buffer, pH 9.5, was added and the ds cDNA was denaturated by heating at
94°C for 5 min
and cooled down. The treated glass slides were prepared as following: Glass
slides were
cleaned overnight in 25% solution of nitric acid at room temperature, washed 3
times by
1 S acetone, treated with 1 % aminopropyl-trimethoxysilane for 3 hrs at room
temperature,
washed two times with acetone, heated at 120°C for 6 hrs and then
treated with 0.2 % of
para-phenylendiisothiocyanate (95:5 acetone-water solution) at room
temperature for 3 hrs,
then washed two times by acetone and dried in vacuum with desiccant. All cDNA
probes
were transferred in 384-well plate and printed on treated glass slides using
384 pin tool and
20 Biomek 2000 (Beckman) robot. After printing, the arrays were incubated in
wet chamber at
37°C overnight, then ultraviolet-cross linked to the surface by
subjecting the slides to 30 mJ
of energy (Stratagene Stratalinker). The arrays were washed with 1 % of sodium
borohydrate
in 0.1 M NaOH, then washed 3 times in distilled water, dried in vacuum and
stored with
desiccant.
Example 7- Hvbridization Cy3 -labeled cDNA Tar~et or Cy3/Cy5 labeled cDNA
targ_etsl with glass cDNA array
1. A solution of ExpressHyb (CLONTECH) and sheared salmon testes DNA (Sigma)
was prepared as follows:
a. ~ ml of ExpressHybTM was prewarmed at 50-60°C.
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b. 0.5 mg of the sheared salmon testes DNA was heated at 95-l00 °C for
5
min, and then chilled quickly on ice.
c. Heat-denatured sheared salmon testes DNA was mixed with prewarmed
ExpressHyb.
2. The glass cDNA array was placed in a hybridization container, and 1 ml of
the
solution prepared in step 1 above was added.
3. Prehybridization was conducted for 5 min with continuous agitation at
65°C.
4. Labeled cDNA probe as prepared in example 3, step C 13, above, (about 200
~cl) was
mixed with 2 ,ul ( 1 ~g/~1) of human Cot- I DNA , and denaturated at
99°C for 2
min.
5. The mixture prepared in Step 4 was added to the hybridization box from Step
3 and
the two solutions were mixed together thoroughly. The container was sealed by
sealing tape.
6. Hybridization was allowed to proceed overnight with continuous agitation at
65°C.
7. The hybridization solution was carefully removed and discarded in an
appropriate
container, and replaced with 10 ml of Wash Solution 1 (2X SSC, 0.1% SDS). The
array was washed for 10 min with continuous agitation at 65°C. The step
was
repeated two times.
8. Additional 10-min washes were performed in 10 ml of Wash Solution 2 (0. 1 X
SSC,
0.1 % SDS) with continuous agitation at 65 °C.
9. Using forceps, the cDNA array was removed from the container, briefly
washed in 0.
1 XSSC and excess buffer was removed from surface by centrifugation in a
Beckman
CS-6R centrifuge at 2000 rpm.
10. Glass arrays were scanned using a custom-built laser scanner equipped by
green (Cy3
chapel) and red ( Cy5 chapel) solid state laser built in UCLA. Images were
scanned
at a resolution of 20 /.em per pixel.
It is evident from the above results and discussion that the subject invention
provides
a rapid, high throughput means to simply and quickly obtain a broad-scale
screening of gene
expression in a variety of different samples. Only simple hybridization
protocols need be
employed with the subject arrays, and signals can be detected using any
convenient and
readily available detection device. Despite their simplicity. assays conducted
with the
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subject arrays yield a large amount of information regarding the expression of
numerous
different and important genes in a particular sample at substantially the same
time, and thus
have use in many different types of applications. including drug discovery and
characterization, disease research, and the (ike.
All publications and patent applications cited in this specification are
herein
incorporated by reference as if each individual publication or patent
application were
specifically and individually indicated to be incorporated by reference. The
citation of any
publication is for its disclosure prior to the filing date and should not be
construed as an
admission that the present invention is not entitled to antedate such
publication by virtue of
prior invention.
Although the foregoing invention has been described in some detail by way of
illustration and example for purposes of clarity of understanding, it is
readily apparent to
those of ordinary skill in the art in light of the teachings of this invention
that certain changes
and modifications may be made thereto without departing from the spirit or
scope of the
appended claims.
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