U.S. patent application number 10/506111 was filed with the patent office on 2006-11-02 for method and nucleic acids for the analysis of a colon cell proliferative disorder.
This patent application is currently assigned to Epigenomics AG. Invention is credited to Peter Adorjan, Evelyne Becker, Matthias Burger, Ralf Lesche, Sabine Maier, Inko Nimmrich, Tamas Rujan, Armin Schmitt.
Application Number | 20060246433 10/506111 |
Document ID | / |
Family ID | 27675653 |
Filed Date | 2006-11-02 |
United States Patent
Application |
20060246433 |
Kind Code |
A1 |
Adorjan; Peter ; et
al. |
November 2, 2006 |
Method and nucleic acids for the analysis of a colon cell
proliferative disorder
Abstract
The present invention relates to modified and genomic sequences,
to oligonucleotides and/or PNA-oligomers for detecting the cytosine
methylation state of genomic DNA, as well as to a method for
ascertaining genetic and/or epigenetic parameters of genes for use
in the differentiation, diagnosis, treatment and/or monitoring of
colon cell proliferative disorders, or the predisposition to colon
cell proliferative disorders.
Inventors: |
Adorjan; Peter; (Berlin,
DE) ; Burger; Matthias; (Berlin, DE) ; Maier;
Sabine; (Berlin, DE) ; Nimmrich; Inko;
(Berlin, DE) ; Becker; Evelyne; (Berlin, DE)
; Lesche; Ralf; (Berlin, DE) ; Rujan; Tamas;
(Berlin, DE) ; Schmitt; Armin; (Breitengupbach,
DK) |
Correspondence
Address: |
DAVIS WRIGHT TREMAINE, LLP
2600 CENTURY SQUARE
1501 FOURTH AVENUE
SEATTLE
WA
98101-1688
US
|
Assignee: |
Epigenomics AG
Kleine Praesidentenstrasse 1
Berlin
DE
10178
|
Family ID: |
27675653 |
Appl. No.: |
10/506111 |
Filed: |
February 27, 2003 |
PCT Filed: |
February 27, 2003 |
PCT NO: |
PCT/EP03/02035 |
371 Date: |
September 16, 2005 |
Current U.S.
Class: |
435/6.12 ;
435/287.2; 435/6.16 |
Current CPC
Class: |
C12Q 1/6886 20130101;
Y10T 436/143333 20150115; A61P 35/00 20180101; C12Q 2600/154
20130101; A61P 43/00 20180101 |
Class at
Publication: |
435/006 ;
435/287.2 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C12M 1/34 20060101 C12M001/34 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 27, 2002 |
EP |
02002551.4 |
Claims
1. A gene panel, comprising at least one target nucleic acid from
the group of genes and/or their regulatory regions comprising MDR1,
APOC2, CACNA1G, EGR4, AR, RB1, GPIb beta, MYOD1, WT1, HLA-F, ELK1,
APC, BCL2, CALCA, CDH1, CDKN1A, CDKN1B (p27 Kip1), CDKN2a, CDKN2B,
CD44, CSPG2, DAPK1, EGFR, EYA4, GSTP1, GTBP/MSH6, HIC-1, HRAS,
IGF2, LKB1, MGMT, MLH1, MNCA9, MSH3, MYC, N33, PAX6, PGR, PTEN,
RARB, SFN, S100A2, TGFBR2, TIMP3, TP53, TP73, VHL, CDKN1C, CAV1,
CDH13, DRG1, PTGS2, THBS1, TPEF (=TMEFF2; =HPP1), DNMT1, CEA, MB,
PCNA, CDC2, ESR1, CASP8, RASSF1, MSH4, MSH5, and wherein at least
one target nucleic acid is selected from the gene CD44 or GPIb beta
and/or their regulatory regions.
2. The gene panel according to claim 1, wherein the target nucleic
acids are selected from the group of genes and/or their regulatory
regions comprising APC, CALCA, CAV1, CD44, CDH1, CDH13, CDKN2a,
CSPG2, DAPK1, EGFR, EGR4, ESR1, GSTP1, GTBP/MSH6, HLA-F, IGF2,
LKB1, MLH1, MYOD1, N33, PTEN, PTGS2, TGFBR2, TP73, TPEF (=TMEFF2;
--HPP1), WT1, and EYA4, and wherein at least one target nucleic
acid is selected from the gene CD44 and/or its regulatory
regions.
3. The gene panel according to claim 1, wherein the target nucleic
acids are selected from the group of genes and/or their regulatory
regions comprising APC, AR, BCL2, CALCA, CAV1, CD44, CDH1, CDH13,
CDKN2a, CEA, CSPG2, DAPK1, EGFR, EGR4, ESR1, GPIb beta, GSTP1,
GTBP/MSH6, HIC-1, HLA-F, IGF2, LKB1, MGMT, MLH1, MSH3, MYC, MYOD1,
N33, PCNA, PGR, PTEN, PTGS2, RARB, RASSF1, S100A2, TGFBR2, TP73,
TPEF (=TMEFF2; =HPP1), WT1, and EYA4, and wherein at least one
target nucleic acid is selected from the gene CD44 and/or its
regulatory regions.
4. The gene panel according to claim 1, wherein the target nucleic
acids are selected from the group of genes and/or their regulatory
regions comprising APC, CAV1, CD44, CDH13, CSPG2, EGFR, GSTP1,
HLA-F, IGF2, N33, PTEN, PTGS2, TP73, TPEF (=TMEFF2; =HPP1), and
EYA4, and wherein at least one target nucleic acid is selected from
the gene CD44 and/or its regulatory regions.
5. The gene panel according to claim 1, wherein said target nucleic
acids are selected from the group of genes and/or their regulatory
regions consisting of GPIb beta and CDKN2a.
6. The gene panel according to any of claims 1 to 5, wherein said
target nucleic acids are selected from the group comprising SEQ ID
NO: 133 to SEQ ID NO: 388 and sequences complementary thereto.
7. The gene panel according to any of claims 1 to 6, wherein said
panel is present in the form of a nucleic acid or peptide nucleic
acid array for the analysis of colon cell proliferative disorders
associated with the methylation state of said target nucleic
acids.
8. The array according to claim 7, characterised in that a solid
phase surface of said array is composed of silicon, glass,
polystyrene, aluminium, steel, iron, copper, nickel, silver, or
gold.
9. An oligonucleotide array, characterised in that said array
comprises at least one oligomer, in particular an oligonucleotide
or peptide nucleic acid (PNA)-oligomer, said oligomer comprising at
least one base sequence of at least 10 nucleotides which hybridises
to or is identical to a pretreated genomic DNA according to one of
the SEQ ID NO: 133 to SEQ ID NO: 388, and wherein at least one
target nucleic acid is selected from the pretreated genomic DNA of
the gene CD44 or GPIb beta and/or their regulatory regions.
10. The oligonucleotide array according to claim 9, wherein the
base sequence of the oligonucleotides includes at least one CpG or
TpG dinucleotide sequence.
11. The oligonucleotide array according to claim 10, wherein the
cytosine of said at least one CpG or TpG dinucleotide is/are
located in the middle third of the oligomer.
12. The oligonucleotide array according to any of claims 9 to 11,
wherein the base sequence of the oligonucleotides is selected from
the group of SEQ ID NO: 519 to SEQ ID NO: 1030.
13. The oligonucleotide array according to any of claims 9 to 12,
comprising a set of oligonucleotides selected from the group of at
least two oligonucleotides according to SEQ ID NO: 986 to 895, at
least two oligonucleotides according to SEQ ID NO: 895 to 906, 909
to 918, 921 to 924, 931, 932, 941, 942, 971, 972, and 987 to 990,
at least two oligonucleotides according to SEQ ID NO: 895 to 954,
957 to 962, 965 to 970, 975 to 978, 981 to 986, and 991 to 1028,
and at least two oligonucleotides according to SEQ ID NO: 1005,
1006, 1029, and 1030.
14. A method for detecting and differentiating between colon cell
proliferative disorders, comprising the following steps; a)
providing a biological sample containing genomic DNA, b) extracting
said genomic DNA, c) converting cytosine bases in said genomic DNA
sample which are unmethylated at the 5-position, by treatment, to
uracil or another base which is dissimilar to cytosine in terms of
base pairing behaviour, d) providing a panel or an array according
to any of claims 1 to 13, and e) identifying the methylation status
of one or more cytosine positions based on said array.
15. The method according to claim 14, characterised in that the
reagent is a solution of bisulfite, hydrogen sulfite or
disulfite.
16. The method according to claim 14 or 15, characterised in that
the fragments of said pretreated genomic DNA are amplified by means
of the polymerase chain reaction CPCR) prior to step (d).
17. The method according to any of claims 14 to 16, characterised
in that more than ten different fragments having a length of
100-2000 base pairs are amplified.
18. The method according to any of claims 14 to 17, characterised
in that the amplification step is carried out using a set of primer
oligonucleotides comprising SEQ ID NO: 389 to SEQ ID NO: 518.
19. The method according to any of claims 14 to 18, characterised
in that the amplification step preferentially amplifies DNA which
is of particular interest in healthy and/or diseased colon tissues,
based on the specific genomic methylation status of colon tissue,
as opposed to background DNA.
20. The method according to any of claims 14 to 19, characterised
in that identifying the methylation status of one or more cytosine
positions based on said array involves a hybridisation of each
amplificate to an oligonucleotide or peptide nucleic acid
(PNA)-oligomer.
21. The method according to claim 20, characterised in that the
amplificates are labelled.
22. The method according to claim 21, further comprising detecting
the amplificates or fragments of the amplificates by mass
spectrometry.
23. A method for detecting and differentiating between colon cell
proliferative disorders, comprising the following steps; a)
providing a biological sample containing genomic DNA, b) extracting
said genomic DNA, c) providing a gene panel according to any of
claims 1 to 6, d) digesting the genomic DNA according to said gene
panel with one or more methylation sensitive restriction enzymes,
and e) detection of the DNA fragments generated in the digest of
step d).
24. The method according to claim 23, wherein the DNA digest is
amplified prior to Step e).
25. The method according to claim 24, wherein the amplification is
carried out by means of the polymerase chain reaction (PCR).
26. The method according to claim 24 or 25, wherein the
amplification of more than one DNA fragments is carried out in one
reaction vessel.
27. The method according to any of claims 14 to 26, wherein said
method differentiates between normal colon tissue and colon cell
proliferative disorder tissue, differentiates between colon adenoma
tissue and normal colon tissue, differentiates between colon
carcinoma tissue and normal colon tissue or differentiates between
colon adenoma tissue and colon carcinoma tissue.
28. A kit comprising a bisulfite (=disulfite, hydrogen sulfite)
reagent as well as oligonucleotides and/or PNA-oligomers comprising
at least one base sequence of at least 10 nucleotides which
hybridises to or is identical to a pretreated genomic DNA according
to one of the SEQ ID NO: 133 to SEQ ID NO: 388, and wherein at
least one base sequence is selected from the pretreated genomic DNA
of the gene CD44 or GPIb beta and/or their regulatory regions.
29. Use of a gene panel according to any of claims 1 to 6 or of an
array according to any of claims 7 to 13 for the detection of a
predisposition to, differentiation between subclasses, diagnosis,
prognosis, treatment, and/or monitoring of colon cell proliferative
disorders.
Description
FIELD OF THE INVENTION
[0001] The levels of observation that have been studied by the
methodological developments of recent years in molecular biology,
are the genes themselves, the translation of these genes into RNA,
and the resulting proteins. The question of which gene is switched
on at which point in the course of the development of an
individual, and how the activation and inhibition of specific genes
in specific cells and tissues are controlled is correlatable to the
degree and character of the methylation of the genes or of the
genome. In this respect, pathogenic conditions may manifest
themselves in a changed methylation pattern of individual genes or
of the genome.
[0002] Colorectal cancer is the fourth leading cause of cancer
mortality in men and women, although ranking third in frequency in
men and second in women. The 5-year survival rate is 61% over all
stages with early detection being a prerequisite for curative
therapy of the disease. Up to 95% of all colorectal cancers are
adenocarcinomas of varying differentiation grades.
[0003] Sporadic colon cancer develops in a multistep process
starting with the pathologic transformation of normal colonic
epithelium to an adenoma which consecutively progresses to invasive
cancer. The progression rate of benign colonic adenomas depends
strongly on their histologic appearance: whereas tubular-type
adenomas tend to progress to malignant tumors very rarely, villous
adenomas, particularly if larger than 2 cm in diameter, have a
significant malignant potential.
[0004] During progression from benign proliferative lesions to
malignant neoplasms several genetic and epigenetic alterations
occur. Somatic mutation of the APC gene seems to be one of the
earliest events in 75 to 80% of colorectal adenomas and carcinomas.
Activation of K-RAS is thought to be a critical step in the
progression towards a malignant phenotype. Consecutively, mutations
in other oncogenes as well as alterations leading to inactivation
of tumour suppressor genes accumulate.
[0005] Aberrant DNA methylation within CpG islands is among the
earliest and most common alterations in human malignancies leading
to abrogation or overexpression of a broad spectrum of genes. In
addition, abnormal methylation has been shown to occur in CpG rich
regulatory elements in intronic and coding parts of genes for
certain tumours. In contrast to the specific hypermethylation of
tumour suppressor genes, an overall hypomethylation of DNA can be
observed in tumour cells. This decrease in global methylation can
be detected early, far before the development of frank tumour
formation. Also, correlation between hypomethylation and increased
gene expression was reported for many oncogenes. In colon cancer,
aberrant DNA methylation constitutes one of the most prominent
alterations and inactivates many tumor suppressor genes such as
p14ARF, p16INK4a, THBS1, MINT2, and MINT31 and DNA mismatch repair
genes such as hMLH1.
[0006] In the molecular evolution of colorectal cancer, DNA
methylation errors have been suggested to play two distinct roles.
In normal colonic mucosa cells, methylation errors accumulate as a
function of age or as time-dependent events predisposing these
cells to neoplastic transformation. For example, hypermethylation
of several loci could be shown to be already present in adenomas,
particularly in the tubulovillous and villous subtype. At later
stages, increased DNA methylation of CpG islands plays an important
role in a subset of tumours affected by the so called CpG island
methylator phenotype (CIMP). Most CIMP+ tumours, which constitute
about 15% of all sporadic colorectal cancers, are characterised by
microsatellite instability (MIN) due to hypermethylation of the
hMLH1 promoter and other DNA mismatch repair genes. By contrast,
CIMP- colon cancers evolve along a more classic genetic instability
pathway (CIN), with a high rate of p53 mutations and chromosomal
changes.
[0007] However, the molecular subtypes do not only show varying
frequencies regarding molecular alterations. According to the
presence of either micro satellite instability or chromosomal
aberrations, colon cancer can be subclassified into two classes,
which also exhibit significant clinical differences. Almost all MIN
tumours originate in the proximal colon (ascending and
transversum), whereas 70% of CIN tumours are located in the distal
colon and rectum. This has been attributed to the varying
prevalence of different carcinogens in different sections of the
colon. Methylating carcinogens, which constitute the prevailing
carcinogen in the proximal colon have been suggested to play a role
in the pathogenesis of MIN cancers, whereas CIN tumours are thought
to be more frequently caused by adduct-forming carcinogens, which
occur more frequently in distal parts of the colon and rectum.
Moreover, MIN tumours have a better prognosis than do tumours with
a CIN phenotype and respond better to adjuvant chemotherapy.
[0008] The identification of markers for the differentiation of
colon carcinoma as well as for early detection are main goals of
current research.
[0009] 5-methylcytosine is the most frequent covalent base
modification in the DNA of eukaryotic cells. It plays a role, for
example, in the regulation of the transcription, in genetic
imprinting, and in tumorigenesis. Therefore, the identification of
5-methylcytosine as a component of genetic information is of
considerable interest. However, 5-methylcytosine positions cannot
be identified by sequencing since 5-methylcytosine has the same
base pairing behaviour as cytosine. Moreover, the epigenetic
information carried by 5-methylcytosine is completely lost during
PCR amplification.
[0010] A relatively new and currently the most frequently used
method for analysing DNA for 5-methylcytosine is based upon the
specific reaction of bisulfite with cytosine which, upon subsequent
alkaline hydrolysis, is converted to uracil which corresponds to
thymidine in its base pairing behaviour. However, 5-methylcytosine
remains unmodified under these conditions. Consequently, the
original DNA is converted in such a manner that methylcytosine,
which originally could not be distinguished from cytosine by its
hybridisation behaviour, can now be detected as the only remaining
cytosine using "normal" molecular biological techniques, for
example, by amplification and hybridisation or sequencing. All of
these techniques are based on base pairing which can now be fully
exploited. In terms of sensitivity, the prior art is defined by a
method which encloses the DNA to be analysed in an agarose matrix,
thus preventing the diffusion and renaturation of the DNA
(bisulfite only reacts with single-stranded DNA), and which
replaces all precipitation and purification steps with fast
dialysis (Olek A, Oswald J, Walter J. A modified and improved
method for bisulphite based cytosine methylation analysis. Nucleic
Acids Res. 1996 Dec. 15;24(24):5064-6). Using this method, it is
possible to analyse individual cells, which illustrates the
potential of the method. However, currently only individual regions
of a length of up to approximately 3000 base pairs are analysed, a
global analysis of cells for thousands of possible methylation
events is not possible. However, this method cannot reliably
analyse very small fragments from small sample quantities either.
These are lost through the matrix in spite of the diffusion
protection.
[0011] An overview of the further known methods of detecting
5-methylcytosine may be gathered from the following review article:
Rein, T., DePamphilis, M. L., Zorbas, H., Nucleic Acids Res. 1998,
26, 2255.
[0012] To date, barring few exceptions (e.g., Zeschnigk M, Lich C,
Buiting K, Doerfler W, Horsthemke B. A single-tube PCR test for the
diagnosis of Angelman and Prader-Willi syndrome based on allelic
methylation differences at the SNRPN locus. Eur J Hum Genet. 1997
March-April;5(2):94-8) the bisulfite technique is only used in
research. Always, however, short, specific fragments of a known
gene are amplified subsequent to a bisulfite treatment and either
completely sequenced (Olek A, Walter J. The pre-implantation
ontogeny of the H19 methylation imprint. Nat Genet. 1997
November;17(3):275-6) or individual cytosine positions are detected
by a primer extension reaction (Gonzalgo M L, Jones P A. Rapid
quantitation of methylation differences at specific sites using
methylation-sensitive single nucleotide primer extension
(Ms-SNuPE). Nucleic Acids Res. 1997 Jun. 15;25(12):2529-31, WO
95/00669) or by enzymatic digestion (Xiong Z, Laird P W. COBRA: a
sensitive and quantitative DNA methylation assay. Nucleic Acids
Res. 1997 Jun. 15;25(12):2532-4). In addition, detection by
hybridisation has also been described (Olek et al., WO
99/28498).
[0013] Further publications dealing with the use of the bisulfite
technique for methylation detection in individual genes are: Grigg
G, Clark S. Sequencing 5-methylcytosine residues in genomic DNA.
Bioessays. 1994 June;16(6):431-6, 431; Zeschnigk M, Schmitz B,
Dittrich B, Buiting K, Horsthemke B, Doerfler W. Imprinted segments
in the human genome: different DNA methylation patterns in the
Prader-Willi/Angelman syndrome region as determined by the genomic
sequencing method. Hum Mol Genet. 1997 March;6(3):387-95; Feil R,
Charlton J, Bird A P, Walter J, Reik W. Methylation analysis on
individual chromosomes: improved protocol for bisulphite genomic
sequencing. Nucleic Acids Res. 1994 Feb. 25;22(4):695-6; Martin V,
Ribieras S, Song-Wang X, Rio M C, Dante R. Genomic sequencing
indicates a correlation between DNA hypomethylation in the 5'
region of the pS2 gene and its expression in human breast cancer
cell lines. Gene. 1995 May 19;157(1-2):261-4; WO 97/46705 and WO
95/15373.
[0014] An overview of the Prior Art in oligomer array manufacturing
can be gathered from a special edition of Nature Genetics (Nature
Genetics Supplement, Volume 21, January 1999), published in January
1999, and from the literature cited therein.
[0015] Fluorescently labelled probes are often used for the
scanning of immobilised DNA arrays. The simple attachment of Cy3
and Cy5 dyes to the 5'-OH of the specific probe are particularly
suitable for fluorescence labels. The detection of the fluorescence
of the hybridised probes may be carried out, for example via a
confocal microscope. Cy3 and Cy5 dyes, besides many others, are
commercially available.
[0016] Matrix Assisted Laser Desorption Ionization Mass
Spectrometry (MALDI-TOF) is a very efficient development for the
analysis of biomolecules (Karas M, Hillenkamp F. Laser desorption
ionisation of proteins with molecular masses exceeding 10,000
daltons. Anal Chem. 1988 Oct. 15;60(20):2299-301). An analyte is
embedded in a light-absorbing matrix. The matrix is evaporated by a
short laser pulse thus transporting the analyte molecule into the
vapour phase in an unfragmented manner. The analyte is ionised by
collisions with matrix molecules. An applied voltage accelerates
the ions into a field-free flight tube. Due to their different
masses, the ions are accelerated at different rates. Smaller ions
reach the detector sooner than bigger ones.
[0017] MALDI-TOF spectrometry is excellently suited to the analysis
of peptides and proteins. The analysis of nucleic acids is somewhat
more difficult (Gut I G, Beck S. DNA and Matrix Assisted Laser
Desorption Ionisation Mass Spectrometry. Current Innovations and
Future Trends. 1995, 1; 147-57). The sensitivity to nucleic acids
is approximately 100 times worse than to peptides and decreases
disproportionally with increasing fragment size. For nucleic acids
having a multiply negatively charged backbone, the ionisation
process via the matrix is considerably less efficient. In MALDI-TOF
spectrometry, the selection of the matrix plays an eminently
important role. For the desorption of peptides, several very
efficient matrixes have been found which produce a very fine
crystallisation. There are now several responsive matrixes for DNA,
however, the difference in sensitivity has not been reduced. The
difference in sensitivity can be reduced by chemically modifying
the DNA in such a manner that it becomes more similar to a peptide.
Phosphorothioate nucleic acids in which the usual phosphates of the
backbone are substituted with thiophosphates can be converted into
a charge-neutral DNA using simple alkylation chemistry (Gut I G,
Beck S. A procedure for selective DNA alkylation and detection by
mass spectrometry. Nucleic Acids Res. 1995 Apr. 25;23(8):1367-73).
The coupling of a charge tag to this modified DNA results in an
increase in sensitivity to the same level as that found for
peptides. A further advantage of charge tagging is the increased
stability of the analysis against impurities which make the
detection of unmodified substrates considerably more difficult.
[0018] Genomic DNA is obtained from DNA of cell, tissue or other
test samples using standard methods. This standard methodology is
found in references such as Sambrook, Fritsch and Maniatis eds.,
Molecular Cloning: A Laboratory Manual, 1989.
DESCRIPTION
[0019] The invention provide a method for the analysis of
biological samples for features associated with the development of
colon cell proliferative disorders, characterised in that the
nucleic acid of at least one member of the group comprising MDR1,
APOC2, CACNA1G, EGR4, AR, RB1, GPIb beta, MYOD1, WT1, HLA-F, ELK1,
APC, BCL2, CALCA, CDH1, CDKN1A, CDKN1B (p27 Kip1), CDKN2a, CDKN2B,
CD44, CSPG2, DAPK1, EGFR, EYA4, GSTP1, GTBP/MSH6, HIC-1, HRAS,
IGF2, LKB1, MGMT, MLH1, MNCA9, MSH3, MYC, N33, PAX6, PGR, PTEN,
RARB, SFN, S100A2, TGFBR2, TIMP3, TP53, TP73, VHL, CDKN1C, CAV1,
CDH13, DRG1, PTGS2, THBS1, TPEF (=TMEFF2; =HPP1), DNMT1, CEA, MB,
PCNA, CDC2, ESR1, CASP8, RASSF1, MSH4, MSH5 is/are contacted with a
reagent or series of reagents capable of distinguishing between
methylated and non methylated CpG dinucleotides within the genomic
sequence of interest.
[0020] The present invention makes available a method for
ascertaining genetic and/or epigenetic parameters of genomic DNA.
The method is for use in the improved diagnosis, treatment and
monitoring of colon cell proliferative disorders, more specifically
by enabling the improved identification of and differentiation
between subclasses of said disorder and the genetic predisposition
to said disorders. The invention presents improvements over the
state of the art in that it enables a highly specific
classification of colon carcinomas, thereby allowing for improved
and informed treatment of patients.
[0021] In a particularly preferred embodiment the present invention
makes available methods and nucleic acids that allow the
differentiation between colon carcinoma, colon adenoma and normal
colon tissue.
[0022] Furthermore, the method enables the analysis of cytosine
methylations and single nucleotide polymorphisms.
[0023] The genes that form the basis of the present invention can
be used to form a "gene panel", i.e. a collection comprising the
particular genetic sequences of the present invention and/or their
respective informative methylation sites. The formation of gene
panels allow for a quick and specific analysis of the disorders
they are related with. The gene panels described in this invention
can be used with surprisingly high efficiency for the diagnosis,
treatment and monitoring of and the analysis of colon cell
proliferative disorders as described herein. The use of multiple
CpG sites from a diverse array of genes, allows for a relatively
high degree of sensitivity and specificity in comparison to single
gene diagnostic and detection tools. Furthermore, the panel as
described herein may be adapted for use in the analysis of many
aspects of colon cell proliferative disorders.
[0024] In a preferred embodiment, the method comprises the
following steps:
[0025] In the first step of the method the genomic DNA sample must
be isolated from tissue or cellular sources. Such sources may
include colon tissue samples, cell lines, histological slides, body
fluids, or tissue embedded in paraffin. Extraction may be by means
that are standard to one skilled in the art, these include the use
of detergent lysates, sonification and vortexing with glass beads.
Once the nucleic acids have been extracted the genomic double
stranded DNA is used in the analysis.
[0026] In a preferred embodiment the DNA may be cleaved prior to
the next step of the method, this may be by any means standard in
the state of the art, in particular, but not limited to, with
restriction endonucleases.
[0027] In the second step of the method, the genomic DNA sample is
treated in such a manner that cytosine bases which are unmethylated
at the 5'-position are converted to uracil, thymine, or another
base which is dissimilar to cytosine in terms of hybridisation
behaviour. This will be understood as `pretreatment`
hereinafter.
[0028] The above described treatment of genomic DNA is preferably
carried out with bisulfite (sulfite, disulfite) and subsequent
alkaline hydrolysis which results in a conversion of non-methylated
cytosine nucleobases to uracil or to another base which is
dissimilar to cytosine in terms of base pairing behaviour. If
bisulfite solution is used for the reaction, then an addition takes
place at the non-methylated cytosine bases. Moreover, a
denaturating reagent or solvent as well as a radical interceptor
must be present. A subsequent alkaline hydrolysis then gives rise
to the conversion of non-methylated cytosine nucleobases to uracil.
The chemically converted DNA is then used for the detection of
methylated cytosines.
[0029] Fragments of the pretreated DNA are amplified, using sets of
primer oligonucleotides according to SEQ ID NO: 389 to SEQ ID NO:
518, and a, preferably heat-stable, polymerase. Because of
statistical and practical considerations, preferably more than ten
different fragments having a length of 100-2000 base pairs are
amplified. The amplification of several DNA segments can be carried
out simultaneously in one and the same reaction vessel. Usually,
the amplification is carried out by means of a polymerase chain
reaction (PCR).
[0030] The method may also be enabled by the use of alternative
primers, the design of such primers is obvious to one skilled in
the art. These should include at least two oligonucleotides whose
sequences are each reverse complementary or identical to an at
least 18 base-pair long segment of the base sequences specified in
the appendix (SEQ ID NO: 133 to SEQ ID NO: 388). Said primer
oligonucleotides are preferably characterised in that they do not
contain any CpG dinucleotides. In a particularly preferred
embodiment of the method, the sequence of said primer
oligonucleotides are designed so as to selectively anneal to and
amplify, only the colon tissue specific DNA of interest, thereby
minimising the amplification of background or non relevant DNA. In
the context of the present invention, background DNA is taken to
mean genomic DNA which does not have a relevant tissue specific
methylation pattern, in this case, the relevant tissue being colon,
both healthy and diseased.
[0031] According to the present invention, it is preferred that at
least one primer oligonucleotide is bound to a solid phase during
amplification. The different oligonucleotide and/or PNA-oligomer
sequences can be arranged on a plane solid phase in the form of a
rectangular or hexagonal lattice, the solid phase surface
preferably being composed of silicon, glass, polystyrene,
aluminium, steel, iron, copper, nickel, silver, or gold, it being
possible for other materials such as nitrocellulose or plastics to
be used as well.
[0032] The fragments obtained by means of the amplification can
carry a directly or indirectly detectable label. Preferred are
labels in the form of fluorescence labels, radionuclides, or
detachable molecule fragments having a typical mass which can be
detected in a mass spectrometer, it being preferred that the
fragments that are produced have a single positive or negative net
charge for better detectability in the mass spectrometer. The
detection may be carried out and visualised by means of matrix
assisted laser desorption/ionisation mass spectrometry (MALDI) or
using electron spray mass spectrometry (ESI).
[0033] The amplificates obtained in the second step of the method
are subsequently hybridised to an array or a set of
oligonucleotides and/or PNA probes. In this context, the
hybridisation preferably takes place in the manner described as
follows. The set of probes used during the hybridisation is
preferably composed of at least 10 oligonucleotides or
PNA-oligomers. However, it is understood and as well claimed, that
the process can be conducted using only one Oligonucleotide or PNA
probe. In the process, the amplificates hybridise to
oligonucleotides previously bonded to a solid phase. In a
particularly preferred embodiment, the oligonucleotides are taken
from the group comprising SEQ ID NO: 519 to SEQ ID NO: 1030. In a
further preferred embodiment the oligonucleotides are taken from
the group comprising SEQ ID NO: 895 to SEQ ID NO: 1030. The
non-hybridised fragments are subsequently removed. Said
oligonucleotides contain at least one base sequence having a length
of 10 nucleotides which is reverse complementary or identical to a
segment of the base sequences specified in the appendix, the
segment containing at least one CpG or TpG dinucleotide. In a
further preferred embodiment the cytosine of the CpG dinucleotide,
or in the case of TpG, the thiamine, is the 5.sup.th to 9.sup.th
nucleotide from the 5'-end of the 10-mer. One oligonucleotide
exists for each CpG or TpG dinucleotide.
[0034] In the fifth step of the method, the non-hybridised
amplificates are removed.
[0035] In the final step of the method, the hybridised amplificates
are detected. In this context, it is preferred that labels attached
to the amplificates are identifiable at each position of the solid
phase at which an oligonucleotide sequence is located.
[0036] According to the present invention, it is preferred that the
labels of the amplificates are fluorescence labels, radionuclides,
or detachable molecule fragments having a typical mass which can be
detected in a mass spectrometer. The mass spectrometer is preferred
for the detection of the amplificates, fragments of the
amplificates or of probes which are complementary to the
amplificates, it being possible for the detection to be carried out
and visualised by means of matrix assisted laser
desorption/ionisation mass spectrometry (MALDI) or using electron
spray mass spectrometry (ESI). The produced fragments may have a
single positive or negative net charge for better detectability in
the mass spectrometer.
[0037] The aforementioned method is preferably used for
ascertaining genetic and/or epigenetic parameters of genomic
DNA.
[0038] In order to enable this method, the invention further
provides the modified DNA of genes MDR1, APOC2, CACNA1G, EGR4, AR,
RB1, GPIb beta, MYOD1, WT1, HLA-F, ELK1, APC, BCL2, CALCA, CDH1,
CDKN1A, CDKN1B (p27 Kip1), CDKN2a, CDKN2B, CD44, CSPG2, DAPK1,
EGFR, EYA4, GSTP1, GTBP/MSH6, HIC-1, HRAS, IGF2, LKB1, MGMT, MLH1,
MNCA9, MSH3, MYC, N33, PAX6, PGR, PTEN, RARB, SFN, S100A2, TGFBR2,
TIMP3, TP53, TP73, VHL, CDKN1C, CAV1, CDH13, DRG1, PTGS2, THBS1,
TPEF (=TMEFF2; =HPP1), DNMT1, CEA, MB, PCNA, CDC2, ESR1, CASP8,
RASSF1, MSH4, MSH5 as well as oligonucleotides and/or PNA-oligomers
for detecting cytosine methylations within said genes. The present
invention is based on the discovery that genetic and epigenetic
parameters and, in particular, the cytosine methylation patterns of
genomic DNA are particularly suitable for improved diagnosis,
treatment and monitoring of colon cell proliferative disorders.
Furthermore, the invention enables the differentiation between
different subclasses of colon cell proliferative disorders or
detection of a predisposition to colon cell proliferative
disorders.
[0039] The nucleic acids according to the present invention can be
used for the analysis of genetic and/or epigenetic parameters of
genomic DNA.
[0040] This objective is achieved according to the present
invention using a nucleic acid containing a sequence of at least 18
bases in length of the pretreated genomic DNA according to one of
SEQ ID NO: 133 to SEQ ID NO: 388 and sequences complementary
thereto.
[0041] The modified nucleic acid could heretofore not be connected
with the ascertainment of disease relevant genetic and epigenetic
parameters.
[0042] The object of the present invention is further achieved by
an oligonucleotide or oligomer for the analysis of pretreated DNA,
for detecting the genomic cytosine methylation state, said
oligonucleotide containing at least one base sequence having a
length of at least 10 nucleotides which hybridises to a pretreated
genomic DNA according to SEQ ID NO: 133 through to SEQ ID NO: 388.
The oligomer probes according to the present invention constitute
important and effective tools which, for the first time, make it
possible to ascertain specific genetic and epigenetic parameters
during the analysis of biological samples for features associated
with the development of colon cell proliferative disorders. Said
oligonucleotides allow the improved diagnosis, treatment and
monitoring of colon cell proliferative disorders and detection of
the predisposition to said disorders. Furthermore, they allow the
differentiation of different subclasses of colon cell proliferative
disorders. The base sequence of the oligomers preferably contains
at least one CpG or TpG dinucleotide. The probes may also exist in
the form of a PNA (peptide nucleic acid) which has particularly
preferred pairing properties. Particularly preferred are
oligonucleotides according to the present invention in which the
cytosine of the CpG dinucleotide is the 5.sup.th-9.sup.th
nucleotide from the 5'-end of the 13-mer; in the case of
PNA-oligomers, it is preferred for the cytosine of the CpG
dinucleotide to be the 4.sup.th-6.sup.th nucleotide from the 5'-end
of the 9-mer.
[0043] The oligomers according to the present invention are
normally used in so called "sets" which contain at least one
oligomer for each of the CpG dinucleotides within SEQ ID NO: 133
through SEQ ID NO: 388. Preferred is a set which contains at least
one oligomer for each of the CpG dinucleotides, from SEQ ID NO: 519
to SEQ ID NO: 1030. Further preferred is a set comprising SEQ ID
NO: 895 to SEQ ID NO: 1030.
[0044] In the case of the sets of oligonucleotides according to the
present invention, it is preferred that at least one
oligonucleotide is bound to a solid phase. It is further preferred
that all the oligonucleotides of one set are bound to a solid
phase.
[0045] The present invention moreover relates to a set of
preferably at least 10 n (oligonucleotides and/or PNA-oligomers)
used for detecting the cytosine methylation state of genomic DNA
using treated versions of said genomic DNA (according to SEQ ID NO:
133 to SEQ ID NO: 388 and sequences complementary thereto).
However, it is understood and as well claimed, that the process can
be conducted using only one Oligonucleotide or PNA oligomer. These
probes enable improved diagnosis, treatment and monitoring of colon
cell proliferative disorders. In particular they enable the
differentiation between different sub classes of colon cell
proliferative disorders and the detection of a predisposition to
said disorders. In a particularly preferred embodiment the set
comprises SEQ ID NO: 519 to SEQ ID NO: 1030.
[0046] The set of oligomers may also be used for detecting single
nucleotide polymorphisms (SNPs) using pretreated genomic DNA
according to one of SEQ ID NO: 133 to SEQ ID NO: 388.
[0047] According to the present invention, it is preferred that an
arrangement of different oligonucleotides and/or PNA-oligomers (a
so-called "array") made available by the present invention is
present in a manner that it is likewise bound to a solid phase.
This array of different oligonucleotide- and/or PNA-oligomer
sequences can be characterised in that it is arranged on the solid
phase in the form of a rectangular or hexagonal lattice. The solid
phase surface is preferably composed of silicon, glass,
polystyrene, aluminium, steel, iron, copper, nickel, silver, or
gold. However, nitrocellulose as well as plastics such as nylon
which can exist in the form of pellets or also as resin matrices
are suitable alternatives.
[0048] Therefore, a further subject matter of the present invention
is a method for manufacturing an array fixed to a carrier material
for the improved diagnosis, treatment and monitoring of colon cell
proliferative disorders, the differentiation between different
subclasses of colon cell proliferative disorders and/or detection
of the predisposition to colon cell proliferative disorders. In
said method at least one oligomer according to the present
invention is coupled to a solid phase. Methods for manufacturing
such arrays are known, for example, from patent U.S. Pat. No.
5,744,305 by means of solid-phase chemistry and photolabile
protecting groups.
[0049] A further subject matter of the present invention relates to
a DNA chip for the improved diagnosis, treatment and monitoring of
colon cell proliferative disorders. Furthermore the DNA chip
enables detection of the predisposition to colon cell proliferative
disorders and the differentiation between different subclasses of
colon cell proliferative disorders. The DNA chip contains at least
one nucleic acid according to the present invention. DNA chips are
known, for example, in patent U.S. Pat. No. 5,837,832.
[0050] Moreover, a subject matter of the present invention is a kit
which may be composed, for example, of a bisulfite-containing
reagent, a set of primer oligonucleotides containing at least two
oligonucleotides whose sequences in each case correspond or are
complementary to a 18 base long segment of the base sequences
specified in the appendix (SEQ ID NO: 133 to SEQ ID NO: 388),
oligonucleotides and/or PNA-oligomers as well as instructions for
carrying out and evaluating the described method. However, a kit
along the lines of the present invention can also contain only part
of the aforementioned components.
[0051] The oligomers according to the present invention or arrays
thereof as well as a kit according to the present invention are
intended to be used for the improved diagnosis, treatment and
monitoring of colon cell proliferative disorders. Furthermore the
use of said inventions extends to the differentiation between
different subclasses of colon cell proliferative disorders and
detection of the predisposition to colon cell proliferative
disorders. According to the present invention, the method is
preferably used for the analysis of important genetic and/or
epigenetic parameters within genomic DNA, in particular for use in
improved diagnosis, treatment and monitoring of colon cell
proliferative disorders, detection of the predisposition to said
disorders and the differentiation between subclasses of said
disorders.
[0052] The methods according to the present invention are used, for
example, for improved diagnosis, treatment and monitoring of colon
cell proliferative disorders progression, detection of the
predisposition to said disorders and the differentiation between
subclasses of said disorders. A further embodiment of the invention
is a method for the analysis of the methylation status of genomic
DNA without the need for pretreatment. In the first step of the
method the genomic DNA sample must be isolated from tissue or
cellular sources. Such sources may include cell lines, histological
slides, body fluids, or tissue embedded in paraffin. Extraction may
be by means that are standard to one skilled in the art, these
include the use of detergent lysates, sonification and vortexing
with glass beads. Once the nucleic acids have been extracted the
genomic double stranded DNA is used in the analysis.
[0053] In a preferred embodiment the DNA may be cleaved prior to
the treatment, this may be any means standard in the state of the
art, in particular with restriction endonucleases. In the second
step, the DNA is then digested with one or more methylation
sensitive restriction enzymes. The digestion is carried out such
that hydrolysis of the DNA at the restriction site is informative
of the methylation status of a specific CpG dinucleotide.
[0054] In the third step the restriction fragments are amplified.
In a preferred embodiment this is carried out using a polymerase
chain reaction.
[0055] In the final step the amplificates are detected. The
detection may be by any means standard in the art, for example, but
not limited to, gel electrophoresis analysis, hybridisation
analysis, incorporation of detectable tags within the PCR products,
DNA array analysis, MALDI or ESI analysis.
[0056] The present invention moreover relates to the diagnosis
and/or prognosis of events which are disadvantageous or relevant to
patients or individuals in which important genetic and/or
epigenetic parameters within genomic DNA, said parameters obtained
by means of the present invention may be compared to another set of
genetic and/or epigenetic parameters, the differences serving as
the basis for the diagnosis and/or prognosis of events which are
disadvantageous or relevant to patients or individuals.
[0057] In the context of the present invention the term
"hybridisation" is to be understood as a bond of an oligonucleotide
to a completely complementary sequence along the lines of the
Watson-Crick base pairings in the sample DNA, forming a duplex
structure.
[0058] In the context of the present invention, "genetic
parameters" are mutations and polymorphisms of genomic DNA and
sequences further required for their regulation. To be designated
as mutations are, in particular, insertions, deletions, point
mutations, inversions and polymorphisms and, particularly
preferred, SNPs (single nucleotide polymorphisms).
[0059] In the context of the present invention "methylation state
analysis" is taken to mean the analysis of cytosines within a
nucleic acid in order to ascertain whether they are methylated or
not. In the context of the present invention, "epigenetic
parameters" are, in particular, cytosine methylations and further
modifications of DNA bases of genomic DNA and sequences further
required for their regulation. Further epigenetic parameters
include, for example, the acetylation of histones which, cannot be
directly analysed using the described method but which, in turn,
correlates with the DNA methylation.
[0060] In the following, the present invention will be explained in
greater detail on the basis of the sequences and examples without
being limited thereto.
[0061] SEQ ID NO: 1 to SEQ ID NO: 64 represent 5' and/or regulatory
regions of the genomic DNA of genes MDR1, APOC2, CACNA1G, EGR4, AR,
RB1, GPIb beta, MYOD1, WT1, HLA-F, ELK1, APC, BCL2, CALCA, CDH1,
CDKN1A, CDKN1B (p27 Kip1), CDKN2a, CDKN2B, CD44, CSPG2, DAPK1,
EGFR, EYA4, GSTP1, GTBP/MSH6, HIC-1, HRAS, IGF2, LKB1, MGMT, MLH1,
MNCA9, MSH3, MYC, N33, PAX6, PGR, PTEN, RARB, SFN, S100A2, TGFBR2,
TIMP3, TP53, TP73, VHL, CDKN1C, CAV1, CDH13, DRG1, PTGS2, THBS1,
TPEF (=TMEFF2; =HPP1), DNMT1, CEA, MB, PCNA, CDC2, ESR1, CASP8,
RASSF1, MSH4, MSH5. These sequences are derived from the ensembl
database (date 01.10.2001) (http://www.ensembl.org) and will be
taken to include all minor variations of the sequence material
which are currently unforeseen, for example, but not limited to,
minor deletions and SNPs.
[0062] SEQ ID 133 to 388 exhibit the pretreated sequences of DNA
derived from genes MDR1, APOC2, CACNA1G, EGR4, AR, RB1, GPIb beta,
MYOD1, WT1, HLA-F, ELK1, APC, BCL2, CALCA, CDH1, CDKN1A, CDKN1B
(p27 Kip1), CDKN2a, CDKN2B, CD44, CSPG2, DAPK1, EGFR, EYA4, GSTP1,
GTBP/MSH6, HIC-1, HRAS, IGF2, LKB1, MGMT, MLH1, MNCA9, MSH3, MYC,
N33, PAX6, PGR, PTEN, RARB, SFN, S100A2, TGFBR2, TIMP3, TP53, TP73,
VHL, CDKN1C, CAV1, CDH13, DRG1, PTGS2, THBS1, TPEF (=TMEFF2;
=HPP1), DNMT1, CEA, MB, PCNA, CDC2, ESR1, CASP8, RASSF1, MSH4,
MSH5. These sequences will be taken to include all minor variations
of the sequence material which are currently unforeseen, for
example, but not limited to, minor deletions and SNPs.
[0063] SEQ ID NO: 389 to SEQ ID NO: 518 exhibit the sequences of
primer oligonucleotides for the amplification of pretreated DNA
according to Sequence ID NO: 133 to SEQ ID NO: 388.
[0064] SEQ ID NO: 65 to SEQ ID NO: 132 exhibit the sequences of
oligomers which are useful for the analysis of CpG positions within
genomic DNA according to SEQ ID NO: 1 to SEQ ID NO: 64.
[0065] SEQ ID NO: 519 to SEQ ID NO: 1030 exhibit the sequences of
oligomers which are useful for the analysis of the methylation
status of CpG positions within genomic DNA according to SEQ ID NO:
1 to SEQ ID NO: 64 after treatment of said genomic DNA with
bisulfite.
[0066] SEQ ID NO: 895 to SEQ ID NO: 1030 exhibit the sequences of
oligomers which are particularly useful for the analysis of CpG
positions within genomic DNA according to SEQ ID NO: 1 to SEQ ID
NO: 64, after treatment of said with bisulfite and are subject to a
preferred embodiment of this invention.
DESCRIPTION OF FIGURES
[0067] FIG. 1: Differentiation between healthy colon tissue and
adenoma or carcinoma colon tissue according to Example 2. The
labels on the left side of the plot are gene and CpG identifiers,
these can be cross referenced using Table 3 and Table 7. The labels
on the right side of the figure give the significance (p-value,
T-test) of the difference between the means of the two groups. Each
row corresponds to a single CpG and each column to the methylation
levels of one sample. CpGs are ordered according to their
contribution to the differentiation between the two tissue types
(A=healthy, B=non healthy) with increasing contribution from top to
bottom. Black indicates total methylation at a given CpG position,
white represents no methylation at the particular position, with
degrees of methylation represented in grey, from light (low
proportion of methylation) to dark (high proportion of
methylation).
[0068] FIG. 2: Differentiation between healthy colon tissue and
carcinoma colon tissue according to Example 2. The labels on the
left side of the plot are gene and CpG identifiers, these can be
cross referenced using Table 4 and Table 7. The labels on the right
side of the figure give the significance (p-value, T-test) of the
difference between the means of the two groups. Each row
corresponds to a single CpG and each column to the methylation
levels of one sample. CpGs are ordered according to their
contribution to the differentiation between the two tissue types
(A=healthy, B=carcinoma) with increasing contribution from top to
bottom. Black indicates total methylation at a given CpG position,
white represents no methylation at the particular position, with
degrees of methylation represented in grey, from light (low
proportion of methylation) to dark (high proportion of
methylation).
[0069] FIG. 3: Differentiation between healthy colon tissue and
adenoma colon tissue according to Example 2. The labels on the left
side of the plot are gene and CpG identifiers, these can be cross
referenced in Table 5 and Table 7. The labels on the right side
give the significance (p-value, T-test) of the difference between
the means of the two groups. Each row corresponds to a single CpG
and each column to the methylation levels of one sample. CpGs are
ordered according to their contribution to the distinction to the
differential diagnosis between the two tissue types (A=healthy,
B=adenoma) with increasing contribution from top to bottom. Black
indicates total methylation at a given CpG position, white
represents no methylation at the particular position, with degrees
of methylation represented in grey, from light (low proportion of
methylation) to dark (high proportion of methylation). Due to
formatting of the page only 40 CpGs are shown in this figure.
[0070] FIG. 4: Differentiation between carcinoma colon tissue and
adenoma colon tissue according to Example 2. The labels on the left
side of the plot are gene and CpG identifiers, these can be cross
referenced in Table 6 and Table 7. The labels on the right side
give the significance (p-value, T-test) of the difference between
the means of the two groups. Each row corresponds to a single CpG
and each column to the methylation levels of one sample. CpGs are
ordered according to their contribution to the distinction to the
differential diagnosis between the two tissue types (A=carcinoma,
B=adenoma) with increasing contribution from top to bottom. Black
indicates total methylation at a given CpG position, white
represents no methylation at the particular position, with degrees
of methylation represented in grey, from light (low proportion of
methylation) to dark (high proportion of methylation).
EXAMPLES 1 AND 2
Digital Phenotype
[0071] In the following examples, multiplex PCR was carried out
upon tissue samples originating from colon adenomas or colon
carcinoma. Multiplex PCR was also carried out upon healthy colon
tissue. Each sample was treated in the manner described below in
Example 1 in order to deduce the methylation status of CpG
positions, the CpG methylation information for each sample was
collated and then used in an analysis, as detailed in Example 2. An
alternative method for the analysis of CpG methylation status is
described in Example 3.
EXAMPLE 1
[0072] In the first step the genomic DNA was isolated from the cell
samples using the Wizzard kit from (Promega).
[0073] The isolated genomic DNA from the samples are treated using
a bisulfite solution hydrogen sulfite, disulfite). The treatment is
such that all non methylated cytosines within the sample are
converted to thiamidine, conversely 5-methylated cytosines within
the sample remain unmodified.
[0074] The treated nucleic acids were then amplified using
multiplex PCRs, amplifying 8 fragments per reaction with Cy5
fluorescently labelled primers. PCR primers used are described in
Table 1. PCR conditions were as follows.
Reaction Solution:
10 ng bisulfite treated DNA
3,5 mM MgCl2
400 .mu.M dNTPs
2 pmol each primer
1 U Hot Star Taq (Qiagen)
[0075] Forty cycles were carried out as follows. Denaturation at
95.degree. C. for 15 min, followed by annealing at 55.degree. C.
for 45 sec., primer elongation at 65.degree. C. for 2 min. A final
elongation at 65.degree. C. was carried out for 10 min.
[0076] All PCR products from each individual sample were then
hybridised to glass slides carrying a pair of immobilised
oligonucleotides for each CpG position under analysis. Each of
these detection oligonucleotides was designed to hybridise to the
bisulphite converted sequence around one CpG site which was either
originally unmethylated (TG) or methylated (CG). See Table 2 for
further details of all hybridisation oligonucleotides used (both
informative and non-informative) Hybridisation conditions were
selected to allow the detection of the single nucleotide
differences between the TG and CG variants.
[0077] 5 .mu.l volume of each multiplex PCR product was diluted in
10.times.Ssarc buffer (10.times.Ssarc:230 ml 20.times.SSC, 180 ml
sodium lauroyl sarcosinate solution 20%, dilute to 1000 ml with
dH2O). The reaction mixture was then hybridised to the detection
oligonucleotides as follows. Denaturation at 95.degree. C., cooling
down to 10.degree. C., hybridisation at 42.degree. C. overnight
followed by washing with 10.times.Ssarc and dH2O at 42.degree.
C.
[0078] Fluorescent signals from each hybridised oligonucleotide
were detected using genepix scanner and software. Ratios for the
two signals (from the CG oligonucleotide and the TG oligonucleotide
used to analyse each CpG position) were calculated based on
comparison of intensity of the fluorescent signals.
EXAMPLE 2
[0079] The data obtained according to Example 1 is then sorted into
a ranked matrix (as shown in FIGS. 1 to 4) according to CpG
methylation differences between the two classes of tissues, using
an algorithm. The most significant CpG positions are at the bottom
of the matrix with significance decreasing towards the top. Black
indicates total methylation at a given CpG position, white
represents no methylation at the particular position, with degrees
of methylation represented in grey, from light (low proportion of
methylation) to dark (high proportion of methylation). Each row
represents one specific CpG position within a gene and each column
shows the methylation profile for the different CpGs for one
sample. On the left side a CpG and gene identifier is shown this
may be cross referenced with the accompanying tables (Table 1 and
7) in order to ascertain the gene in question and the detection
oligomer used. On the right side p values for the individual CpG
positions are shown. The p values are the probabilities that the
observed distribution occurred by chance in the data set.
[0080] For selected distinctions, we trained a learning algorithm
(support vector machine, SVM). The SVM (as discussed by F. Model,
P. Adorjan, A. Olek, C. Piepenbrock, Feature selection for DNA
methylation based cancer classification. Bioinformatics. 2001
June;17 Suppl 1:S157-64) constructs an optimal discriminant between
two classes of given training samples. In this case each sample is
described by the methylation patterns (CG/TG ratios) at the
investigated CpG sites. The SVM was trained on a subset of samples
of each class, which were presented with the diagnosis attached.
Independent test samples, which were not shown to the SVM before
were then presented to evaluate, if the diagnosis can be predicted
correctly based on the predictor created in the training round.
This procedure was repeated several times using different
partitions of the samples, a method called cross-validation. Please
note that all rounds are performed without using any knowledge
obtained in the previous runs. The number of correct
classifications was averaged over all runs, which gives a good
estimate of our test accuracy (percent of correct classified
samples over all rounds).
Healthy Colon Tissue Compared to Non Healthy Colon Tissue (Colon
Adenoma and Colon Carcinoma) (FIG. 1)
[0081] FIG. 1 shows the differentiation of healthy tissue from non
healthy tissue wherein the non healthy specimens are obtained from
either colon adenoma or colon carcinoma tissue. The evaluation is
carried out using informative CpG positions from 27 genes.
Informative CpG positions are further described in Table 3.
Healthy Colon Tissue Compared to Colon Carcinoma Tissue (FIG.
2)
[0082] FIG. 2 shows the differentiation of healthy tissue from
carcinoma tissue using informative CpG positions from 15 genes.
Informative CpG positions are further described in Table 4.
Healthy Colon Tissue Compared to Colon Adenoma Tissue (FIG. 3)
[0083] FIG. 3 shows the differentiation of healthy tissue from
adenoma tissue using informative CpG positions from 40 genes.
Informative CpG positions are further described in Table 5.
Colon Carcinoma Tissue Compared to Colon Adenoma Tissue (FIG.
4)
[0084] FIG. 4 shows the differentiation of carcinoma tissue from
adenoma tissue using informative CpG positions from 2 genes.
Informative CpG positions are further described in Table 6.
EXAMPLE 3
Identification of the Methylation Status of a CpG Site Within the
Gene CD44.
[0085] A fragment of the bisulfite treated DNA of the gene CD44
(Seq ID NO: 20) was PCR amplified using primers
GAAAGGAGAGGTTAAAGGTTG (Seq ID NO 429) and AACTCACTTAACTCCAATCCC
(Seq ID NO 430). The resultant fragment (696 bp in length)
contained an informative CpG at position 235. The amplificate DNA
was digested with the restriction endonuclease Apa I, recognition
site GGGCC. Hydrolysis by said endonuclease is blocked by
methylation of the CpG at position 235 of the amplificate. The
digest was used as a control.
[0086] Genomic DNA was isolated from sample using the DNA wizzard
DNA isolation kit (Promega). Each sample was digested using Apa I
according to manufacturer's recommendations (New England
Biolabs).
[0087] 10 ng of each genomic digest was then amplified using PCR
primers GAAAGGAGAGGTTAAAGGTTG and AACTCACTTAACTCCAATCCC. The PCR
reactions were performed using a thermocycler (Eppendorf GmbH)
using 10 ng of DNA, 6 pmol of each primer, 200 .mu.M of each dNTP,
1.5 mM MgCl2 and 1 U of HotstartTaq (Qiagen AG). The other
conditions were as recommended by the Taq polymerase manufacturer.
Using the above mentioned primers, gene fragments were amplified by
PCR performing a first denaturation step for 14 min at 96.degree.
C., followed by 30-45 cycles (step 2: 60 sec at 96.degree. C., step
3: 45 sec at 52.degree. C., step 4: 75 sec at 72.degree. C.) and a
subsequent final elongation of 10 min at 72.degree. C. The presence
of PCR products was analysed by agarose gel electrophoresis.
[0088] PCR products were detectable with Apa I hydrolysed DNA
isolated wherein the CpG position in question was up-methylated,
when step 2 to step 4 of the cycle program were repeated 34, 37,
39, 42 and 45 fold. In contrast PCR products were only detectable
with Apa I hydrolysed DNA isolated from down-methylated DNA (and
control DNA) when step 2 to step 4 of the cycle program were
repeated 42 and 45 fold. These results were incorporated into a CpG
methylation matrix analysis as described in Example 2.
[0089] Tables TABLE-US-00001 TABLE 1 PCR primers and products
Amplificate No: Gene: Primer: length: 1 MDR1
TAAGTATGTTGAAGAAAGATTATTGTAG 633 (SEQ ID NO: 1) (SEQ ID NO: 389)
TAAAAACTATCCCATAATAACTCCCAAC (SEQ ID NO: 390) 2 APOC2
ATGAGTAGAAGAGGTGATAT 533 (SEQ ID NO: 2) (SEQ ID NO: 391)
CCCTAAATCCCTTTCTTACC (SEQ ID NO: 392) 3 CACNA1G
GGGATTTAAGAGAAATTGAGGTA 707 (SEQ ID NO: 3) (SEQ ID NO: 393)
AAACCCCAAACATCCTTTAT (SEQ ID NO: 394) 4 EGR4 AGGGGGATTGAGTGTTAAGT
293 (SEQ ID NO: 4) (SEQ ID NO: 395) CCCAAACATAAACACAAAAT (SEQ ID
NO: 396) 5 AR GTAGTAGTAGTAGTAAGAGA 460 (SEQ ID NO: 5) (SEQ ID NO:
397) ACCCCCTAAATAATTATCCT (SEQ ID NO: 398) 6 RB1
TTTAAGTTTGTTTTTGTTTTGGT 718 (SEQ ID NO: 6) (SEQ ID NO: 399)
TCCTACTCTAAATCCTCCTCAA (SEQ ID NO: 400) 7 GPIb beta
GGTGATAGGAGAATAATGTTGG 379 (SEQ ID NO: 7) (SEQ ID NO: 401)
TCTCCCAACTACAACCAAAC (SEQ ID NO: 402) 8 MYOD1
ATTAGGGGTATAGAGGAGTATTGA 883 (SEQ ID NO: 8) (SEQ ID NO: 403)
CTTACAAACCCACAATAAACAA (SEQ ID NO: 404) 9 WT1 AAAGGGAAATTAAGTGTTGT
747 (SEQ ID NO: 9) (SEQ ID NO: 405) TAACTACCCTCAACTTCCC (SEQ ID NO:
406) 10 HLA-F TTGTTGTTTTTAGGGGTTTTGG 946 (SEQ ID NO: 10) (SEQ ID
NO: 407) TCCTTCCCATTCTCCAAATATC (SEQ ID NO: 408) 11 ELK1
AAGTGTTTTAGTTTTTAATGGGTA 966 (SEQ ID NO: 11) (SEQ ID NO: 409)
CAAACCCAAAACTCACCTAT (SEQ ID NO: 410) 12 APC TCAACTACCATCAACTTCCTTA
491 (SEQ ID NO: 12) (SEQ ID NO: 411) AATTTATTTTTAGTGTTGTAGTGGG (SEQ
ID NO: 412) 13 BCL2 GTATTTTATGTTAAGGGGGAAA 640 (SEQ ID NO: 13) (SEQ
ID NO: 413) AAAAACCACAATCCTCCC (SEQ ID NO: 414) 14 CALCA
GTTTTGGAAGTATGAGGGTG 614 (SEQ ID NO: 14) (SEQ ID NO: 415)
CCAAATTCTAAACCAATTTCC (SEQ ID NO: 416) 15 CDH1 GAGGTTGGGGTTAGAGGAT
478 (SEQ ID NO: 15) (SEQ ID NO: 417) CAAACTCACAAATACTTTACAATTC (SEQ
ID NO: 418) 16 CDKN1A GGATTAGTGGGAATAGAGGTG 408 (SEQ ID NO: 16)
(SEQ ID NO: 419) AAACCCAAACTCCTAACTACC (SEQ ID NO: 420) 17 CDKN1B
GTGGGGAGGTAGTTGAAGA 478 (p27 Kip1) (SEQ ID NO: 421) (SEQ ID NO: 17)
ATACACCCCTAACCCAAAAT (SEQ ID NO: 422) 18 CDKN2a
TTGAAAATTAAGGGTTGAGG 598 (SEQ ID NO: 18) (SEQ ID NO: 423)
CACCCTCTAATAACCAACCA (SEQ ID NO: 424) 19 CDKN2a
GGGGTTGGTTGGTTATTAGA 256 (SEQ ID NO: 18) (SEQ ID NO: 425)
AACCCTCTACCCACCTAAAT (SEQ ID NO: 426) 20 CDKN2B
GGTTGGTTGAAGGAATAGAAAT 708 (SEQ ID NO: 19) (SEQ ID NO: 427)
CCCACTAAACATACCCTTATTC (SEQ ID NO: 428) 21 CD44
GAAAGGAGAGGTTAAAGGTTG 696 (SEQ ID NO: 20) (SEQ ID NO: 429)
AACTCACTTAACTCCAATCCC (SEQ ID NO: 430) 22 CSPG2
GGATAGGAGTTGGGATTAAGAT 414 (SEQ ID NO: 21) (SEQ ID NO: 431)
AAATCTTTTTCAACACCAAAAT (SEQ ID NO: 432) 23 DAPK1
AACCCTTTCTTCAAATTACAAA 348 (SEQ ID NO: 22) (SEQ ID NO: 433)
TGATTGGGTTTTAGGGAAATA (SEQ ID NO: 434) 24 EGFR
GGGTTTGGTTGTAATATGGATT 732 (SEQ ID NO: 23) (SEQ ID NO: 435)
CCCAACACTACCCCTCTAA (SEQ ID NO: 436) 25 EYA4 GGAAGAGGTGATTAAATGGAT
226 (SEQ ID NO: 24) (SEQ ID NO: 437) CCCAAAAATCAAACAACAA (SEQ ID
NO: 438) 26 GSTP1 ATTTGGGAAAGAGGGAAAG 300 (SEQ ID NO: 25) (SEQ ID
NO: 439) TAAAAACTCTAAACCCCATCC (SEQ ID NO: 440) 27 GTBP/MSH6
CCCTACCCACCAATATACC 278 (SEQ ID NO: 26) (SEQ ID NO: 441)
AGATTGGGGAAGAAGTTGTA (SEQ ID NO: 442) 28 HIC-1 TGGGTTGGAGAAGAAGTTTA
280 (SEQ ID NO: 27) (SEQ ID NO: 443) TCATATTTCCAAAAACACACC (SEQ ID
NO: 444) 29 HRAS CTTATTCCCATCTAAACCCTATT 331 (SEQ ID NO: 28) (SEQ
ID NO: 445) GTGGTTTTGTGAAGTTTTAGGT (SEQ ID NO: 446) 30 IGF2
CCCTTCCCCTTAACTAAACT 364 (SEQ ID NO: 29) (SEQ ID NO: 447)
AATTTGGGTTAGGTTTGGA (SEQ ID NO: 448) 31 LKB1 TAAAAGAAGGATTTTTGATTGG
528 (SEQ ID NO: 30) (SEQ ID NO: 449) CATCTTATTTACCTCCCTCCC (SEQ ID
NO: 450) 32 MGMT AAGGTTTTAGGGAAGAGTGTTT 636 (SEQ ID NO: 31) (SEQ ID
NO: 451) ACCTTTTCCTATCACAAAAATAA (SEQ ID NO: 452) 33 MLH1
TAAGGGGAGAGGAGGAGTTT 545 (SEQ ID NO: 32) (SEQ ID NO: 453)
ACCAATTCTCAATCATCTCTTT (SEQ ID NO: 454) 34 MNCA9
GGGAAGTAGGTTAGGGTTAGTT 616 (SEQ ID NO: 33) (SEQ ID NO: 455)
AAATCCTCCTCTCCAAATAAAT (SEQ ID NO: 456) 35 MSH3 TGTTTGGGATTGGGTAGG
211 (SEQ ID NO: 34) (SEQ ID NO: 457) CATAACCTTTACCTATCTCCTCA (SEQ
ID NO: 458) 36 MYC AGAGGGAGTAAAAGAAAATGGT 712 (SEQ ID NO: 35) (SEQ
ID NO: 459) CCAAATAAACAAAATAACCTCC (SEQ ID NO: 460) 37 N33
TTTTAGATTGAGGTTTTAGGGT 497 (SEQ ID NO: 36) (SEQ ID NO: 461)
ATCCATTCTACCTCCTTTTTCT (SEQ ID NO: 462) 38 PAX6 GGAGGGGAGAGGGTTATG
374 (SEQ ID NO: 37) (SEQ ID NO: 463) TACTATACACACCCCAAAACAA (SEQ ID
NO: 464) 39 PGR TTTTGGGAATGGGTTGTAT 369 (SEQ ID NO: 38) (SEQ ID NO:
465) CTACCCTTAACCTCCATCCTA (SEQ ID NO: 466) 40 PTEN
TTTTAGGTAGTTATATTGGGTATGTT 346 (SEQ ID NO: 39) (SEQ ID NO: 467)
TCAACTCTCAAACTTCCATCA (SEQ ID NO: 468) 41 RARB
TTGTTGGGAGTTTTTAAGTTTT 353 (SEQ ID NO: 40) (SEQ ID NO: 469)
CAAATTCTCCTTCCAAATAAAT (SEQ ID NO: 470) 42 SFN GAAGAGAGGAGAGGGAGGTA
489 (SEQ ID NO: 41) (SEQ ID NO: 471) CTATCCAACAAACCCAACA (SEQ ID
NO: 472) 43 S100A2 GTTTTTAAGTTGGAGAAGAGGA 460 (SEQ ID NO: 42) (SEQ
ID NO: 473) ACCTATAAATCACAACCCACTC (SEQ ID NO: 474) 44 TGFBR2
GTAATTTGAAGAAAGTTGAGGG 296 (SEQ ID NO: 43) (SEQ ID NO: 475)
CCAACAACTAAACAAAACCTCT (SEQ ID NO: 476) 45 TIMP3
TGAGAAAATTGTTGTTTGAAGT 306 (SEQ ID NO: 44) (SEQ ID NO: 477)
CAAAATACCCTAAAAACCACTC (SEQ ID NO: 478) 46 TP53
GGAGTTGTATTGTTGGGAGA 279 (SEQ ID NO: 45) (SEQ ID NO: 479)
TAAAACCCCAATTTTCACTAA (SEQ ID NO: 480) 47 TP73
AGTAAATAGTGGGTGAGTTATGAA 607 (SEQ ID NO: 46) (SEQ ID NO: 481)
GAAAAACCTCTAAAAACTACTCTCC (SEQ ID NO: 482) 48 VHL
TGTAAAATGAATAAAGTTAATGAGTG 362 (SEQ ID NO: 47) (SEQ ID NO: 483)
TCCTAAATTCAAATAATCCTCCT (SEQ ID NO: 484) 49 CDKN1C
GGGGAGGTAGATATTTGGATAA 300 (SEQ ID NO: 48) (SEQ ID NO: 485)
AACTACACCATTTATATTCCCAC
(SEQ ID NO: 486) 50 CAV1 GTTAGTATGTTTGGGGGTAAAT 435 (SEQ ID NO: 49)
(SEQ ID NO: 487) ATAAATAACACCTTCCACCCTA (SEQ ID NO: 488) 51 CDH13
TTTGTATTAGGTTGGAAGTGGT 286 (SEQ ID NO: 50) (SEQ ID NO: 489)
CCCAAATAAATCAACAACAACA (SEQ ID NO: 490) 52 DRG1
GGTTTTGGGTTTAGTGGTAAAT 416 (SEQ ID NO: 51) (SEQ ID NO: 491)
AACTTTCATAACTCACCCTTTC (SEQ ID NO: 492) 53 PTGS2
GATTTTTGGAGAGGAAGTTAAG 381 (SEQ ID NO: 52) (SEQ ID NO: 493)
AAAACTAAAAACCAAACCCATA (SEQ ID NO: 494) 54 THBS1
TGGGGTTAGTTTAGGATAGG 398 (SEQ ID NO: 53) (SEQ ID NO: 495)
CTTAAAAACACTAAAACTTCTCAAA (SEQ ID NO: 496) 55 TPEF
TTGTTTGGGTTAATAAATGGA 295 =TEMFF2; =HPP1) (SEQ ID NO: 497) (SEQ ID
NO: 54) CTTCTCTCTTCTCCCCTCTC (SEQ ID NO: 498) 56 DNMT1
TCCCCATCACACCTAAAA 210 (SEQ ID NO: 55) (SEQ ID NO: 499)
GGGAGGAGGGGATGTATT (SEQ ID NO: 500) 57 CEA TATGGGAGGAGGTTAGTAAGTG
680 (SEQ ID NO: 56) (SEQ ID NO: 501) CCCCAAATCCTACATATAAAAA (SEQ ID
NO: 502) 58 MB GTTTTTGGTAAAGGGGTAGAA 598 (SEQ ID NO: 57) (SEQ ID
NO: 503) CCTAAAATATCAACCTCCACCT (SEQ ID NO: 504) 59 PCNA
TTTTTAGGTTGTAAGGAGGTTTT 608 (SEQ ID NO: 58) (SEQ ID NO: 505)
TAAATACCTCCAACACCTTTCT (SEQ ID NO: 506) 60 CDC2
ATTAGAAGTGAAAGTAATGGAATTT 418 (SEQ ID NO: 59) (SEQ ID NO: 507)
TCAATTTCCAAAAACCAAC (SEQ ID NO: 508) 61 ESR1 AGGGGGAATTAAATAGAAAGAG
662 (SEQ ID NO: 60) (SEQ ID NO: 509) CAATAAAACCATCCCAAATACT (SEQ ID
NO: 510) 62 CASP8 AGTGGATTTGGAGTTTAGATGT 431 (SEQ ID NO: 61) (SEQ
ID NO: 511) AACAAAATAAAAACTTCTCCCA (SEQ ID NO: 512) 63 RASSF1
ACCTCTCTACAAATTACAAATTCA 347 (SEQ ID NO: 62) (SEQ ID NO: 513)
AGTTTGGGTTAGTTTGGGTT (SEQ ID NO: 514) 64 MSH4 AGGATGTTGAGGTTTGAGATT
339 (SEQ ID NO: 63) (SEQ ID NO: 515) CACTATAATAACCACCACCCA (SEQ ID
NO: 516) 65 MSH5 TATTAGGAATAAAGTTGGGGAG 395 (SEQ ID NO: 64) (SEQ ID
NO: 517) AACCCTTCAAACAAAAATAAAA (SEQ ID NO: 518)
[0090] TABLE-US-00002 TABLE 2 Hybridisation oligonucleotides No:
Gene Oligo: 1 MDR1 TTGGTGGTCGTTTTAAGG (SEQ ID NO: 1) (SEQ ID NO:
519) 2 MDR1 TTGGTGGTTGTTTTAAGG (SEQ ID NO: 1) (SEQ ID NO: 520) 3
MDR1 TTGAAAGACGTGTTTATA (SEQ ID NO: 1) (SEQ ID NO: 521) 4 MDR1
TTGAAAGATGTGTTTATA (SEQ ID NO: 1) (SEQ ID NO: 522) 5 MDR1
AGGTGTAACGGAAGTTAG (SEQ ID NO: 1) (SEQ ID NO: 523) 6 MDR1
AGGTGTAATGGAAGTTAG (SEQ ID NO: 1) (SEQ ID NO: 524) 7 MDR1
TAGTTTTTCGAGGAATTA (SEQ ID NO: 1) (SEQ ID NO: 525) 8 MDR1
TAGTTTTTTGAGGAATTA (SEQ ID NO: 1) (SEQ ID NO: 526) 9 APOC2
GAGAGTTTCGTTTTTGTT (SEQ ID NO: 2) (SEQ ID NO: 527) 10 APOC2
GAGAGTTTTGTTTTTGTT (SEQ ID NO: 2) (SEQ ID NO: 528) 11 APOC2
TTGGGGGACGTTATTGTT (SEQ ID NO: 2) (SEQ ID NO: 529) 12 APOC2
TTGGGGGATGTTATTGTT (SEQ ID NO: 2) (SEQ ID NO: 530) 13 APOC2
TGTGTTCGTTCGGAGTTG (SEQ ID NO: 2) (SEQ ID NO: 531) 14 APOC2
TGTGTTTGTTTGGAGTTG (SEQ ID NO: 2) (SEQ ID NO: 532) 15 APOC2
TGGGTTTGCGGAGAATGG (SEQ ID NO: 2) (SEQ ID NO: 533) 16 APOC2
TGGGTTTGTGGAGAATGG (SEQ ID NO: 2) (SEQ ID NO: 534) 17 CACNA1G
TTTAGGAGCGTTAATGTG (SEQ ID NO: 3) (SEQ ID NO: 535) 18 CACNA1G
TTTAGGAGTGTTAATGTG (SEQ ID NO: 3) (SEQ ID NO: 536) 19 CACNA1G
TAGGGTTACGAGGTTAGG (SEQ ID NO: 3) (SEQ ID NO: 537) 20 CACNA1G
TAGGGTTATGAGGTTAGG (SEQ ID NO: 3) (SEQ ID NO: 538) 21 CACNA1G
GGAGGTTACGTTTAGATT (SEQ ID NO: 3) (SEQ ID NO: 539) 22 CACNA1G
GGAGGTTATGTTTAGATT (SEQ ID NO: 3) (SEQ ID NO: 540) 23 CAGNA1G
TTAGGGGTCGTGGATAAA (SEQ ID NO: 3) (SEQ ID NO: 541) 24 CACNA1G
TTAGGGGTTGTGGATAAA (SEQ ID NO: 3) (SEQ ID NO: 542) 25 EGR4
GGTGGGAAGCGTATTTAT (SEQ ID NO: 4) (SEQ ID NO: 543) 26 EGR4
GGTGGGAAGTGTATTTAT (SEQ ID NO: 4) (SEQ ID NO: 544) 27 EGR4
TTATAGTTCGAGTTTTTT (SEQ ID NO: 4) (SEQ ID NO: 545) 28 EGR4
TTATAGTTTGAGTTTTTT (SEQ ID NO: 4) (SEQ ID NO: 546) 29 EGR4
GGAGTTTTCGGTATATAT (SEQ ID NO: 4) (SEQ ID NO: 927) 30 EGR4
GGAGTTTTTGGTATATAT (SEQ ID NO: 4) (SEQ ID NO: 928) 31 AR
TGTTATTTCGAGAGAGGT (SEQ ID NO: 5) (SEQ ID NO: 547) 32 AR
TGTTATTTTGAGAGAGGT (SEQ ID NO: 5) (SEQ ID NO: 548) 33 AR
AGAGGTTGCGTTTTAGAG (SEQ ID NO: 5) (SEQ ID NO: 1027) 34 AR
AGAGGTTGTGTTTTAGAG (SEQ ID NO: 5) (SEQ ID NO: 1028) 35 AR
ATTTTGAGCGAGGTTAGT (SEQ ID NO: 5) (SEQ ID NO: 549) 36 AR
ATTTTGAGTGAGGTTAGT (SEQ ID NO: 5) (SEQ ID NO: 550) 37 AR
GTAGTATTCGAAGGTAGT (SEQ ID NO: 5) (SEQ ID NO: 551) 38 AR
GTAGTATTTGAAGGTAGT (SEQ ID NO: 5) (SEQ ID NO: 552) 39 RB1
TTAGATTTCGGGATAGGG (SEQ ID NO: 6) (SEQ ID NO: 553) 40 RB1
TTAGATTTTGGGATAGGG (SEQ ID NO: 6) (SEQ ID NO: 554) 41 RB1
TATAGTTTCGTTAAGTGT (SEQ ID NO: 6) (SEQ ID NO: 555) 42 RB1
TATAGTTTTGTTAAGTGT (SEQ ID NO: 6) (SEQ ID NO: 556) 43 RB1
GTGTATTTCGGTTTGGAG (SEQ ID NO: 6) (SEQ ID NO: 557) 44 RB1
GTGTATTTTGGTTTGGAG (SEQ ID NO: 6) (SEQ ID NO: 558) 45 RB1
TGGATTTACGTTAGGTTT (SEQ ID NO: 6) (SEQ ID NO: 559) 46 RB1
TGGATTTATGTTAGGTTT (SEQ ID NO: 6) (SEQ ID NO: 560) 47 GPIb beta
TGTTATTTGTCGTTGTAG (SEQ ID NO: 7) (SEQ ID NO: 561) 48 GPIb beta
TGTTATTTGTTGTTGTAG (SEQ ID NO: 7) (SEQ ID NO: 562) 49 GPIb beta
GTGGGAGCGGAAGTTTGA (SEQ ID NO: 7) (SEQ ID NO: 563) 50 GPIb beta
GTGGGAGTGGAAGTTTGA (SEQ ID NO: 7) (SEQ ID NO: 564) 51 GPIb beta
TAGAGTAAGTCGGGTTGT (SEQ ID NO: 7) (SEQ ID NO: 565) 52 GPIb beta
TAGAGTAAGTCGGGTTGTT (SEQ ID NO: 7) (SEQ ID NO: 566) 53 GPIb beta
GGTTAGGTCGTAGTATTG (SEQ ID NO: 7) (SEQ ID NO: 567) 54 GPIb beta
GGTTAGGTTGTAGTATTG (SEQ ID NO: 7) (SEQ ID NO: 568) 55 GPIb beta
GGAGTTCGGTCGGGTTTT (SEQ ID NO: 7) (SEQ ID NO: 1005) 56 GPIb beta
GGAGTTTGGTTGGGTTTT (SEQ ID NO: 7) (SEQ ID NO: 1006) 57 MYOD1
ATAGTAGTCGGGTGTTGG (SEQ ID NO: 8) (SEQ ID NO: 569) 58 MYOD1
ATAGTAGTTGGGTGTTGG (SEQ ID NO: 8) (SEQ ID NO: 570) 59 MYOD1
GTGTTAGTCGTTTAGGGT (SEQ ID NO: 8) (SEQ ID NO: 1009) 60 MYOD1
GTGTTAGTTGTTTAGGGT (SEQ ID NO: 8) (SEQ ID NO: 1010) 61 MYOD1
TAGTTGTTCGTTTGGGTT (SEQ ID NO: 8) (SEQ ID NO: 571) 62 MYOD1
TAGTTGTTTGTTTGGGTT (SEQ ID NO: 8) (SEQ ID NO: 572) 63 MYOD1
AATTAGGTCGGATAGGAG (SEQ ID NO: 8) (SEQ ID NO: 975) 64 MYOD1
AATTAGGTTGGATAGGAG (SEQ ID NO: 8) (SEQ ID NO: 976) 65 WT1
TAGTGAGACGAGGTTTTT (SEQ ID NO: 9) (SEQ ID NO: 1017) 66 WT1
TAGTGAGATGAGGTTTTT (SEQ ID NO: 9) (SEQ ID NO. 1018) 67 WT1
TATATTGGCGAAGGTTAA (SEQ ID NO: 9) (SEQ ID NO: 967) 68 WT1
TATATTGGTGAAGGTTAA (SEQ ID NO: 9) (SEQ ID NO: 968) 69 WT1
TGTTATATCGGTTAGTTG (SEQ ID NO: 9) (SEQ ID NO: 959) 70 WT1
TGTTATATTGGTTAGTTG (SEQ ID NO: 9) (SEQ ID NO: 960) 71 WT1
TTTAGTTTCGATTTTTGG (SEQ ID NO: 9) (SEQ ID NO: 573) 72 WT1
TTTAGTTTTGATTTTTGG (SEQ ID NO: 9) (SEQ ID NO: 574) 73 HLA-F
ATAGGGTACGTTAAGGTT (SEQ ID NO: 10) (SEQ ID NO: 575) 74 HLA-F
ATAGGGTATGTTAAGGTT (SEQ ID NO: 10) (SEQ ID NO: 576) 75 HLA-F
TATTTGGGCGGGTGAGTG (SEQ ID NO: 10) (SEQ ID NO: 939) 76 HLA-F
TATTTGGGTGGGTGAGTG (SEQ ID NO: 10) (SEQ ID NO: 940) 77 HLA-F
GAGAGAAACGGTTTTTGT (SEQ ID NO: 10) (SEQ ID NO: 577) 78 HLA-F
GAGAGAAATGGTTTTTGT (SEQ ID NO: 10) (SEQ ID NO: 578) 79 HLA-F
AGTTGTTTCGTAGATATT (SEQ ID NO: 10) (SEQ ID NO: 989) 80 HLA-F
AGTTGTTTTGTAGATATT (SEQ ID NO: 10) (SEQ ID NO: 990) 81 ELK1
TGTTTAATCGTAGAGTTG (SEQ ID NO: 11) (SEQ ID NO: 579) 82 ELK1
TGTTTAATTGTAGAGTTG
(SEQ ID NO: 11) (SEQ ID NO: 580) 83 ELK1 TTTGTTTTCGTTGAGTAG (SEQ ID
NO: 11) (SEQ ID NO: 581) 84 ELK1 TTTGTTTTTGTTGAGTAG (SEQ ID NO: 11)
(SEQ ID NO: 582) 85 ELK1 GAAGGGTTCGTTTTTTAA (SEQ ID NO: 11) (SEQ ID
NO: 583) 86 ELK1 GAAGGGTTTGTTTTTTAA (SEQ ID NO: 11) (SEQ ID NO:
584) 87 ELK1 ATTAATAGCGTTTTGGTT (SEQ ID NO: 11) (SEQ ID NO: 585) 88
ELK1 ATTAATAGTGTTTTGGTT (SEQ ID NO: 11) (SEQ ID NO: 586) 89 APC
TTTAATCGTATAGTTTGT (SEQ ID NO: 12) (SEQ ID NO: 971) 90 APC
TTTAATTGTATAGTTTGT (SEQ ID NO: 12) (SEQ ID NO: 972) 91 APC
TATTTAGCGGATTATATA (SEQ ID NO: 12) (SEQ ID NO. 587) 92 APC
TATTTAGTGGATTATATA (SEQ ID NO: 12) (SEQ ID NO: 588) 93 APC
TATTTTGGCGGGTTGTAT (SEQ ID NO: 12) (SEQ ID NO: 985) 94 APC
TATTTTGGTGGGTTGTAT (SEQ ID NO: 12) (SEQ ID NO: 986) 95 APC
AAGGTTATCGGTTTAAGA (SEQ ID NO: 12) (SEQ ID NO: 589) 96 APC
AAGGTTATTGGTTTAAGA (SEQ ID NO: 12) (SEQ ID NO: 590) 97 APC
GGGGGACGACGTTTTTGT (SEQ ID NO: 12) (SEQ ID NO: 591) 98 APC
GGGGGATGATGTTTTTGT (SEQ ID NO: 12) (SEQ ID NO: 592) 99 BCL2
AGTGTTTCGCGTGATTGA (SEQ ID NO: 13) (SEQ ID NO: 593) 100 BCL2
AGTGTTTCGCGTGATTGA (SEQ ID NO: 13) (SEQ ID NO: 594) 101 BCL2
TAAGTTGTCGTAGAGGGG (SEQ ID NO: 13) (SEQ ID NO: 595) 102 BCL2
TAAGTTGTTGTAGAGGGG (SEQ ID NO: 13) (SEQ ID NO: 596) 103 BCL2
GGATTTCGTCGTTGTAGA (SEQ ID NO: 13) (SEQ ID NO: 597) 104 BCL2
GGATTTTGTTGTTGTAGA (SEQ ID NO: 13) (SEQ ID NO: 598) 105 BCL2
TTTTGTTACGGTGGTGGA (SEQ ID NO: 13) (SEQ ID NO: 1025) 106 BCL2
TTTTGTTATGGTGGTGGA (SEQ ID NO: 13) (SEQ ID NO: 1026) 107 CALCA
GAGGGTGACGTAATTTAG (SEQ ID NO: 14) (SEQ ID NO: 599) 108 CALCA
GAGGGTGATGTAATTTAG (SEQ ID NO: 14) (SEQ ID NO: 600) 109 CALCA
TGTATTGGCGGAATTTTT (SEQ ID NO: 14) (SEQ ID NO: 601) 110 CALCA
TGTATTGGTGGAATTTTT (SEQ ID NO: 14) (SEQ ID NO: 602) 111 CALCA
ATTAGGTTCGTGTTTTAG (SEQ ID NO: 14) (SEQ ID NO: 953) 112 CALCA
ATTAGGTTTGTGTTTTAG (SEQ ID NO: 14) (SEQ ID NO: 954) 113 CALCA
GTTAGTTTCGGGATATTT (SEQ ID NO: 14) (SEQ ID NO: 603) 114 CALCA
GTTAGTTTTGGGATATTT (SEQ ID NO: 14) (SEQ ID NO: 604) 115 CDH1
TAGAGGATCGTTTGAGTT (SEQ ID NO: 15) (SEQ ID NO: 605) 116 CDH1
TAGAGGATTGTTTGAGTT (SEQ ID NO: 15) (SEQ ID NO: 606) 117 CDH1
GTTGTGATCGTATTATTG (SEQ ID NO: 15) (SEQ ID NO: 607) 118 CDH1
GTTGTGATTGTATTATTG (SEQ ID NO: 15) (SEQ ID NO: 608) 119 CDH1
TTGGGATTCGAATTTAGT (SEQ ID NO: 15) (SEQ ID NO: 609) 120 CDH1
TTGGGATTTGAATTTAGT (SEQ ID NO: 15) (SEQ ID NO: 610) 121 CDH1
AGGGTTATCGCGTTTATG (SEQ ID NO: 15) (SEQ ID NO: 983) 122 CDH1
AGGGTTATTGTGTTTATG (SEQ ID NO: 15) (SEQ ID NO: 984) 123 CDH1
TAGTGGCGTCGGAATTGT (SEQ ID NO: 15) (SEQ ID NO: 929) 124 CDH1
TAGTGGTGTTGGAATTGT (SEQ ID NO: 15) (SEQ ID NO: 930) 125 CDKN1A
AGGTGTATCGTTTTTATA (SEQ ID NO: 16) (SEQ ID NO: 611) 126 CDKN1A
AGGTGTATTGTTTTTATA (SEQ ID NO: 16) (SEQ ID NO: 612) 127 CDKN1A
TGGGTTAGCGGTGAGTTA (SEQ ID NO: 16) (SEQ ID NO: 613) 128 CDKN1A
TGGGTTAGTGGTGAGTTA (SEQ ID NO: 16) (SEQ ID NO: 614) 129 CDKN1A
GTTTATTTCGTGGGGAAA (SEQ ID NO: 16) (SEQ ID NO: 615) 130 CDKN1A
GTTTATTTTGTGGGGAAA (SEQ ID NO: 16) (SEQ ID NO: 616) 131 CDKN1A
TTGGAATTCGGTTAGGTT (SEQ ID NO: 16) (SEQ ID NO: 617) 132 CDKN1A
TTGGAATTTGGTTAGGTT (SEQ ID NO: 16) (SEQ ID NO: 618) 133 CDKN1B (p27
AAGAGAAACGTTGGAATA Kip1) (SEQ ID NO: 619) (SEQ ID NO: 17) 134
CDKN1B (p27 AAGAGAAATGTTGGAATA Kip1) (SEQ ID NO: 620) (SEQ ID NO:
17) 135 CDKN1B (p27 TTTGATTTCGAGGGGAGT Kip1) (SEQ ID NO: 621) (SEQ
ID NO: 17) 136 CDKN1B (p27 TTTGATTTTGAGGGGAGT Kip1) (SEQ ID NO:
622) (SEQ ID NO: 17) 137 CDKN1B (p27 GTATTTGGCGGTTGGATT Kip1) (SEQ
ID NO: 623) (SEQ ID NO: 17) 138 CDKN1B (p27 GTATTTGGTGGTTGGATT
Kip1) (SEQ ID NO: 624) (SEQ ID NO: 17) 139 CDKN1B (p27
TATAATTTCGGGAAAGAA Kip1) (SEQ ID NO: 625) (SEQ ID NO: 17) 140
CDKN1B (p27 TATAATTTTGGGAAAGAA Kip1) (SEQ ID NO: 626) (SEQ ID NO:
17) 141 CDKN2a AGAGTGAACGTATTTAAA (SEQ ID NO: 18) (SEQ ID NO: 627)
142 CDKN2a AGAGTGAATGTATTTAAA (SEQ ID NO: 18) (SEQ ID NO: 628) 143
CDKN2a GTTGTTTTCGGTTGGTGT (SEQ ID NO: 18) (SEQ ID NO: 1029) 144
CDKN2a GTTGTTTTTGGTTGGTGT (SEQ ID NO: 18) (SEQ ID NO: 1030) 145
CDKN2a GATAGGGTCGGAGGGGGT (SEQ ID NO: 18) (SEQ ID NO: 629) 146
CDKN2a GATAGGGTTGGAGGGGGT (SEQ ID NO: 18) (SEQ ID NO: 630) 147
CDKN2a GGAGTTTTCGGTTGATTG (SEQ ID NO: 18) (SEQ ID NO: 997) 148
CDKN2a GGAGTTTTTGGTTGATTG (SEQ ID NO: 18) (SEQ ID NO: 998) 149
CDKN2a AATAGTTACGGTCGGAGG (SEQ ID NO: 18) (SEQ ID NO: 981) 150
CDKN2a AATAGTTATGGTTGGAGG (SEQ ID NO: 18) (SEQ ID NO: 982) 151
CDKN2B ATATTTAGCGAGTAGTGT (SEQ ID NO: 19) (SEQ ID NO: 631) 152
CDKN2B ATAGGGGGCGGAGTTTAA (SEQ ID NO: 19) (SEQ ID NO: 632) 153
CDKN2B ATAGGGGGCGGAGTTTAA (SEQ ID NO: 19) (SEQ ID NO: 633) 154
CDKN2B ATAGGGGGTGGAGTTTAA (SEQ ID NO: 19) (SEQ ID NO: 634) 155
CDKN2B TTATTGTACGGGGTTTTA (SEQ ID NO: 19) (SEQ ID NO: 635) 156
CDKN2B TTATTGTATGGGGTTTTA (SEQ ID NO: 19) (SEQ ID NO: 636) 157
CDKN2B TTTTAAGTCGTAGAAGGA (SEQ ID NO: 19) (SEQ ID NO: 637) 158
CDKN2B TTTTAAGTTGTAGAAGGA (SEQ ID NO: 19) (SEQ ID NO: 638) 159 CD44
GTGGGGTTCGGAGGTATA (SEQ ID NO: 20) (SEQ ID NO: 919) 160 CD44
GTGGGGTTTGGAGGTATA (SEQ ID NO: 20) (SEQ ID NO: 920) 161 CD44
GGTAGTTTCGATTATTTA (SEQ ID NO: 20) (SEQ ID NO: 639) 162 CD44
GGTAGTTTTGATTATTTA (SEQ ID NO: 20) (SEQ ID NO: 640) 163 CD44
TTGTTTAGCGGATTTTAG
(SEQ ID NO: 20) (SEQ ID NO: 897) 164 CD44 TTGTTTAGTGGATTTTAG (SEQ
ID NO: 20) (SEQ ID NO: 898) 165 CD44 TGGTGGTACGTAGTTTGG (SEQ ID NO:
20) (SEQ ID NO: 641) 166 CD44 TGGTGGTATGTAGTTTGG (SEQ ID NO: 20)
(SEQ ID NO: 642) 167 CSPG2 AAGATTTTCGGTTAGTTT (SEQ ID NO: 21) (SEQ
ID NO: 963) 168 CSPG2 AAGATTTTTGGTTAGTTT (SEQ ID NO: 21) (SEQ ID
NO: 964) 169 CSPG2 ATGTGATTCGTTTGGGTA (SEQ ID NO: 21) (SEQ ID NO:
643) 170 CSPG2 ATGTGATTTGTTTGGGTA (SEQ ID NO: 21) (SEQ ID NO: 644)
171 CSPG2 GGGTAACGTCGAATTTAG (SEQ ID NO: 21) (SEQ ID NO: 901) 172
CSPG2 GGGTAATGTTGAATTTAG (SEQ ID NO: 21) (SEQ ID NO: 902) 173 CSPG2
AAAAATTCGCGAGTTTAG (SEQ ID NO: 21) (SEQ ID NO: 945) 174 CSPG2
AAAAATTTGTGAGTTTAG (SEQ ID NO: 21) (SEQ ID NO: 946) 175 DAPK1
GTTGGAGTCGAGGTTTGA (SEQ ID NO: 22) (SEQ ID NO: 645) 176 DAPK1
GTTGGAGTTGAGGTTTGA (SEQ ID NO: 22) (SEQ ID NO: 646) 177 DAPK1
TTTTTTGTCGGATTGGTG (SEQ ID NO: 22) (SEQ ID NO: 647) 178 DAPK1
TTTTTTGTTGGATTGGTG (SEQ ID NO: 22) (SEQ ID NO: 648) 179 DAPK1
GAAGGGAGCGTATTTTAT (SEQ ID NO: 22) (SEQ ID NO: 955) 180 DAPK1
GAAGGGAGTGTATTTTAT (SEQ ID NO: 22) (SEQ ID NO: 956) 181 DAPK1
TTGTTTTTCGGAAATTTG (SEQ ID NO: 22) (SEQ ID NO: 935) 182 DAPK1
TTGTTTTTTGGAAATTTG (SEQ ID NO: 22) (SEQ ID NO: 936) 183 EGRF
TTTGTATTCGGAGTTGGG (SEQ ID NO: 23) (SEQ ID NO: 961) 184 EGRF
TTTGTATTTGGAGTTGGG (SEQ ID NO: 23) (SEQ ID NO: 962) 185 EGRF
GATGATTTCGAGGGTGTT (SEQ ID NO: 23) (SEQ ID NO: 649) 186 EGRF
GATGATTTTGAGGGTGTT (SEQ ID NO: 23) (SEQ ID NO: 650) 187 EGRF
GAGGGTTTCGTAGTGTTG (SEQ ID NO: 23) (SEQ ID NO: 651) 188 EGRF
GAGGGTTTTGTAGTGTTG (SEQ ID NO: 23) (SEQ ID NO: 652) 189 EGRF
TGGGGATTCGAATAAAGG (SEQ ID NO: 23) (SEQ ID NO: 653) 190 EGRF
TGGGGATTTGAATAAAGG (SEQ ID NO: 23) (SEQ ID NO: 654) 191 EGRF
ATTTGGTTCGATTTGGAT (SEQ ID NO: 23) (SEQ ID NO: 931) 192 EGRF
ATTTGGTTTGATTTGGAT (SEQ ID NO: 23) (SEQ ID NO: 932) 193 EYA4
TATATATACGTGTGGGTA (SEQ ID NO: 24) (SEQ ID NO: 655) 194 EYA4
TATATATATGTGTGGGTA (SEQ ID NO: 24) (SEQ ID NO: 656) 195 EYA4
AGTGTATGCGTAGAAGGT (SEQ ID NO: 24) (SEQ ID NO: 923) 196 EYA4
AGTGTATGTGTAGAAGGT (SEQ ID NO: 24) (SEQ ID NO: 924) 197 EYA4
TTTAGATACGAAATGTTA (SEQ ID NO: 24) (SEQ ID NO: 657) 198 EYA4
TTTAGATATGAAATGTTA (SEQ ID NO: 24) (SEQ ID NO: 658) 199 EYA4
AAGTAAGTCGTTGTTGTT (SEQ ID NO: 24) (SEQ ID NO: 921) 200 EYA4
AAGTAAGTTGTTGTTGTT (SEQ ID NO: 24) (SEQ ID NO: 922) 201 GSTP1
GGTTTTTTCGGTTAGTTG (SEQ ID NO: 25) (SEQ ID NO: 659) 202 GSTP1
GGTTTTTTTGGTTAGTTG (SEQ ID NO: 25) (SEQ ID NO: 660) 203 GSTP1
GGAGTTCGCGGGATTTTT (SEQ ID NO: 25) (SEQ ID NO: 905) 204 GSTP1
GGAGTTTGTGGGATTTTT (SEQ ID NO: 25) (SEQ ID NO: 906) 205 GSTP1
GTAGTTTTCGTTATTAGT (SEQ ID NO: 25) (SEQ ID NO: 661) 206 GSTP1
GTAGTTTTTGTTATTAGT (SEQ ID NO: 25) (SEQ ID NO: 662) 207 GTBP/MSH6
GAGGAATTCGGGTTTTAG (SEQ ID NO: 26) (SEQ ID NO: 951) 208 GTBP/MSH6
GAGGAATTTGGGTTTTAG (SEQ ID NO: 26) (SEQ ID NO: 952) 209 GTBP/MSH6
TTTGTTGGCGGGAAATTT (SEQ ID NO: 26) (SEQ ID NO: 925) 210 GTBP/MSH6
TTTGTTGGTGGGAAATTT (SEQ ID NO: 26) (SEQ ID NO: 926) 211 GTBP/MSH6
TTTTGTCGGACGGAGTTT (SEQ ID NO: 26) (SEQ ID NO: 663) 212 GTBP/MSH6
TTTTGTTGGATGGAGTTT (SEQ ID NO: 26) (SEQ ID NO: 664) 213 GTBP/MSH6
AAGGTTTAATCGTTTTGT (SEQ ID NO: 26) (SEQ ID NO: 665) 214 GTBP/MSH6
AAGGTTTAATTGTTTTGT (SEQ ID NO: 26) (SEQ ID NO: 666) 215 HIC-1
TTAAAACGGCGTATAGGG (SEQ ID NO: 27) (SEQ ID NO: 667) 216 HIC-1
TTAAAATGGTGTATAGGG (SEQ ID NO: 27) (SEQ ID NO: 668) 217 HIC-1
AGGAGATTCGAAAGTTTA (SEQ ID NO: 27) (SEQ ID NO: 669) 218 HIC-1
AGGAGATTTGAAAGTTTA (SEQ ID NO: 27) (SEQ ID NO: 670) 219 HIC-1
TTTTAGAGCGTTAGGGTT (SEQ ID NO: 27) (SEQ ID NO: 1021) 220 HIC-1
TTTTAGAGTGTTAGGGTT (SEQ ID NO: 27) (SEQ ID NO: 1022) 221 HRAS
ATAGTGGGCGTAATTGGT (SEQ ID NO: 28) (SEQ ID NO: 671) 222 HRAS
ATAGTGGGTGTAATTGGT (SEQ ID NO: 28) (SEQ ID NO: 672) 223 HRAS
AAATTGGACGTTTAGTTG (SEQ ID NO: 28) (SEQ ID NO: 673) 224 HRAS
AAATTGGATGTTTAGTTG (SEQ ID NO: 28) (SEQ ID NO: 674) 225 HRAS
TAGAAGTCGAGAGATTTG (SEQ ID NO: 28) (SEQ ID NO: 675) 226 HRAS
TAGAAGTTGAGAGATTTG (SEQ ID NO: 28) (SEQ ID NO: 676) 227 HRAS
GAATATTTCGAAGTTTGT (SEQ ID NO: 28) (SEQ ID NO: 677) 228 HRAS
GAATATTTTGAAGTTTGT (SEQ ID NO: 28) (SEQ ID NO: 678) 229 IGF2
AGTTTGAACGATGTAAGA (SEQ ID NO: 29) (SEQ ID NO: 973) 230 IGF2
AGTTTGAATGATGTAAGA (SEQ ID NO: 29) (SEQ ID NO: 974) 231 IGF2
GGTTATTACGATAATTTG (SEQ ID NO: 29) (SEQ ID NO: 679) 232 IGF2
GGTTATTATGATAATTTG (SEQ ID NO: 29) (SEQ ID NO: 680) 233 IGF2
TTGTATGGTCGAGTTTAT (SEQ ID NO: 29) (SEQ ID NO: 941) 234 IGF2
TTGTATGGTTGAGTTTAT (SEQ ID NO: 29) (SEQ ID NO: 942) 235 IGF2
GATTAGGGCGGGAAATAT (SEQ ID NO: 29) (SEQ ID NO: 937) 236 IGF2
GATTAGGGTGGGAAATAT (SEQ ID NO: 29) (SEQ ID NO: 938) 237 IGF2
TGGAGTTTACGGAGGTTT (SEQ ID NO: 29) (SEQ ID NO: 681) 238 IGF2
TGGAGTTTATGGAGGTTT (SEQ ID NO: 29) (SEQ ID NO: 682) 239 LKB1
TTAATTAACGGGTGGGTA (SEQ ID NO: 30) (SEQ ID NO: 683) 240 LKB1
TTAATTAATGGGTGGGTA (SEQ ID NO: 30) (SEQ ID NO: 684) 241 LKB1
TTTAGGTTCGTAAGTTTA (SEQ ID NO: 30) (SEQ ID NO: 965) 242 LKB1
TTTAGGTTTGTAAGTTTA (SEQ ID NO: 30) (SEQ ID NO: 966) 243 LKB1
AGGGAGGTCGTTGGTATT (SEQ ID NO: 30) (SEQ ID NO: 933) 244 LKB1
AGGGAGGTTGTTGGTATT (SEQ ID NO: 30) (SEQ ID NO: 934) 245 MGMT
TAAGGATACGAGTTATAT (SEQ ID NO: 31) (SEQ ID NO: 685) 246 MGMT
TAAGGATATGAGTTATAT (SEQ ID NO: 31) (SEQ ID NO: 686)
247 MGMT TTGGAGAGCGGTTGAGTT (SEQ ID NO: 31) (SEQ ID NO: 687) 248
MGMT TTGGAGAGTGGTTGAGTT (SEQ ID NO: 31) (SEQ ID NO: 688) 249 MGMT
TAGGTTATCGGTGATTGT (SEQ ID NO: 31) (SEQ ID NO: 689) 250 MGMT
TAGGTTATTGGTGATTGT (SEQ ID NO: 31) (SEQ ID NO: 690) 251 MGMT
TAGGGGAGCGGTTTTAGG (SEQ ID NO: 31) (SEQ ID NO: 691) 252 MGMT
TAGGGGAGTGGTTTTAGG (SEQ ID NO: 31) (SEQ ID NO: 692) 253 MGMT
AGTAGGATCGGGATTTTT (SEQ ID NO: 31) (SEQ ID NO: 1001) 254 MGMT
AGTAGGATTGGGATTTTT (SEQ ID NO: 31) (SEQ ID NO: 1002) 255 MLH1
TTGAGAAGCGTTAAGTAT (SEQ ID NO: 32) (SEQ ID NO: 693) 256 MLH1
TTGAGAAGTGTTAAGTAT (SEQ ID NO: 32) (SEQ ID NO: 694) 257 MLH1
TTAGGTAGCGGGTAGTAG (SEQ ID NO: 32) (SEQ ID NO: 949) 258 MLH1
TTAGGTAGTGGGTAGTAG (SEQ ID NO: 32) (SEQ ID NO: 950) 259 MLH1
GTAGTAGTCGTTTTAGGG (SEQ ID NO: 32) (SEQ ID NO: 695) 260 MLH1
GTAGTAGTTGTTTTAGGG (SEQ ID NO: 32) (SEQ ID NO: 696) 261 MLH1
ATAGTTGTCGTTGAAGGG (SEQ ID NO: 32) (SEQ ID NO: 697) 262 MLH1
ATAGTTGTTGTTGAAGGG (SEQ ID NO: 32) (SEQ ID NO: 698) 263 MLH1
TTGGATGGCGTAAGTTAT (SEQ ID NO: 32) (SEQ ID NO: 699) 264 MLH1
TTGGATGGTGTAAGTTAT (SEQ ID NO: 32) (SEQ ID NO: 700) 265 MNCA9
TAAAAGGGCGTTTTGTGA (SEQ ID NO: 33) (SEQ ID NO: 701) 266 MNCA9
TAAAAGGGTGTTTTGTGA (SEQ ID NO: 33) (SEQ ID NO: 702) 267 MNCA9
TAGTTAGTCGTATGGTTT (SEQ ID NO: 33) (SEQ ID NO: 703) 268 MNCA9
TAGTTAGTTGTATGGTTT (SEQ ID NO: 33) (SEQ ID NO: 704) 269 MNCA9
GATTTATTCGGAGAGGAG (SEQ ID NO: 33) (SEQ ID NO: 705) 270 MNCA9
GATTTATTTGGAGAAGAG (SEQ ID NO: 33) (SEQ ID NO: 706) 271 MSH3
ATTTTTCGTTCGATGATA (SEQ ID NO: 34) (SEQ ID NO: 707) 272 MSH3
ATTTTTTGTTTGATGATA (SEQ ID NO: 34) (SEQ ID NO: 708) 273 MSH3
AGTTTAGTCGGGGTTATA (SEQ ID NO: 34) (SEQ ID NO: 709) 274 MSH3
AGTTTAGTTGGGGTTATA (SEQ ID NO: 34) (SEQ ID NO: 710) 275 MSH3
GGGTGAAGCGTTGAGGTT (SEQ ID NO: 34) (SEQ ID NO: 711) 276 MSH3
GGGTGAAGTGTTGAGGTT (SEQ ID NO: 34) (SEQ ID NO: 712) 277 MSH3
AGTATTTTCGTTTGAGGA (SEQ ID NO:34) (SEQ ID NO:1015) 278 MSH3
AGTATTTTTGTTTGAGGA (SEQ ID NO: 34) (SEQ ID NO: 1016) 279 MYC
TTAGAGTGTTCGGTTGTT (SEQ ID NO: 35) (SEQ ID NO: 713) 280 MYC
TTAGAGTGTTTGGTTGTT (SEQ ID NO: 35) (SEQ ID NO: 714) 281 MYC
TTATAATGCGAGGGTTTG (SEQ ID NO: 35) (SEQ ID NO: 1019) 282 MYC
TTATAATGTGAGGGTTTG (SEQ ID NO: 35) (SEQ ID NO: 1020) 283 MYC
AGGATTTTCGAGTTGTGT (SEQ ID NO: 35) (SEQ ID NO: 715) 284 MYC
AGGATTTTTGAGTTGTGT (SEQ ID NO: 35) (SEQ ID NO: 716) 285 MYC
AATTTTAGCGAGAGGTAG (SEQ ID NO: 35) (SEQ ID NO: 717) 286 MYC
AATTTTAGTGAGAGGTAG (SEQ ID NO: 35) (SEQ ID NO: 718) 287 N33
TTGGTTCGGGAAAGGTAA (SEQ ID NO: 36) (SEQ ID NO: 977) 288 N33
TTGGTTTGGGAAAGGTAA (SEQ ID NO: 36) (SEQ ID NO: 978) 289 N33
TGTTATTTCGGAGGGTTT (SEQ ID NO: 36) (SEQ ID NO: 909) 290 N33
TGTTATTTTGGAGGGTTT (SEQ ID NO: 36) (SEQ ID NO: 910) 291 N33
GTTTAGTTAGCGGGTTTT (SEQ ID NO: 36) (SEQ ID NO: 943) 292 N33
GTTTAGTTAGTGGGTTTT (SEQ ID NO: 36) (SEQ ID NO: 944) 293 N33
ATTTAGTTCGGGGGAGGA (SEQ ID NO: 36) (SEQ ID NO: 993) 294 N33
ATTTAGTTTGGGGGAGGA (SEQ ID NO: 36) (SEQ ID NO: 994) 295 PAX6
TATTGTTTCGGTTGTTAG (SEQ ID NO: 37) (SEQ ID NO: 719) 296 PAX6
TATTGTTTTGGTTGTTAG (SEQ ID NO: 37) (SEQ ID NO: 720) 297 PAX6
GTTAGTAGCGAGTTTAGG (SEQ ID NO: 37) (SEQ ID NO: 721) 298 PAX6
GTTAGTAGTGAGTTTAGG (SEQ ID NO: 37) (SEQ ID NO: 722) 299 PAX6
AGAGTTTAGCGTATTTTT (SEQ ID NO: 37) (SEQ ID NO: 723) 300 PAX6
AGAGTTTAGTGTATTTTT (SEQ ID NO: 37) (SEQ ID NO: 724) 301 PGR
GAATTTAGCGAGGGATTG (SEQ ID NO: 38) (SEQ ID NO: 725) 302 PGR
GAATTTAGTGAGGGATTG (SEQ ID NO: 38) (SEQ ID NO: 726) 303 PGR
AGTATGTACGAGTTTGAT (SEQ ID NO: 38) (SEQ ID NO: 727) 304 PGR
AGTATGTATGAGTTTGAT (SEQ ID NO: 38) (SEQ ID NO: 728) 305 PGR
TTAAGTGTCGGATTTGTG (SEQ ID NO: 38) (SEQ ID NO: 1011) 306 PGR
TTAAGTGTTGGATTTGTG (SEQ ID NO: 38) (SEQ ID NO: 1012) 307 PGR
GGGATAAACGATAGTTAT (SEQ ID NO: 38) (SEQ ID NO: 729) 308 PGR
GGGATAAATGATAGTTAT (SEQ ID NO: 38) (SEQ ID NO: 730) 309 PTEN
AGAGTTTGCGGTTTGGGG (SEQ ID NO: 39) (SEQ ID NO: 731) 310 PTEN
AGAGTTTGTGGTTTGGGGT (SEQ ID NO: 39) (SEQ ID NO: 732) 311 PTEN
ATTTTGCGTTCGTATTTA (SEQ ID NO: 39) (SEQ ID NO: 987) 312 PTEN
ATTTTGTGTTTGTATTTA (SEQ ID NO: 39) (SEQ ID NO: 988) 313 PTEN
AGAGTTATCGTTTTGTTT (SEQ ID NO: 39) (SEQ ID NO: 957) 314 PTEN
AGAGTTATTGTTTTGTTT (SEQ ID NO: 39) (SEQ ID NO: 958) 315 PTEN
TGATGTGGCGGGATTTTT (SEQ ID NO: 39) (SEQ ID NO: 947) 316 PTEN
TGATGTGGTGGGATTTTT (SEQ ID NO: 39) (SEQ ID NO: 948) 317 RARB
TAGTAGTTCGGGTAGGGT (SEQ ID NO: 40) (SEQ ID NO: 991) 318 RARB
TAGTAGTTTGGGTAGGGT (SEQ ID NO: 40) (SEQ ID NO: 992) 319 RARB
GGGTTTATCGAAAGTTTA (SEQ ID NO: 40) (SEQ ID NO: 733) 320 RARB
GGGTTTATTGAAAGTTTA (SEQ ID NO: 40) (SEQ ID NO: 734) 321 RARB
AGTTTATTCGTATATATT (SEQ ID NO: 40) (SEQ ID NO: 735) 322 RARB
AGTTTATTTGTATATATT (SEQ ID NO: 40) (SEQ ID NO: 736) 323 RARB
TTTTTATGCGAGTTGTTT (SEQ ID NO: 40) (SEQ ID NO: 737) 324 RARB
TTTTTATGTGAGTTGTTT (SEQ ID NO: 40) (SEQ ID NO: 738) 325 SFN
ATAGAGTTCGGTATTGGT (SEQ ID NO: 41) (SEQ ID NO: 739) 326 SFN
ATAGAGTTTGGTATTGGT (SEQ ID NO: 41) (SEQ ID NO: 740) 327 SFN
GTAGGTCGAACGTTATGA (SEQ ID NO: 41) (SEQ ID NO: 741) 328 SFN
GTAGGTTGAATGTTATGA (SEQ ID NO: 41) (SEQ ID NO: 742) 329 SFN
AAAAGTAACGAGGAGGGT (SEQ ID NO: 41) (SEQ ID NO: 743) 330 SFN
AAAAGTAATGAGGAGGGT (SEQ ID NO: 41) (SEQ ID NO: 744)
331 S100A2 TTTAATTGCGGTTGTGTG (SEQ ID NO: 42) (SEQ ID NO: 745) 332
S100A2 TTTAATTGTGGTTGTGTG (SEQ ID NO: 42) (SEQ ID NO: 746) 333
S100A2 TATATAGGCGTATGTATG (SEQ ID NO: 42) (SEQ ID NO: 747) 334
S100A2 TATATAGGTGTATGTATG (SEQ ID NO: 42) (SEQ ID NO: 748) 335
S100A2 TATGTATACGAGTATTGG (SEQ ID NO: 42) (SEQ ID NO: 999) 336
S100A2 TATGTATATGAGTATTGG (SEQ ID NO: 42) (SEQ ID NO: 1000) 337
S100A2 AGTTTTAGCGTGTGTTTA (SEQ ID NO: 42) (SEQ ID NO: 749) 338
S100A2 AGTTTTAGTGTGTGTTTA (SEQ ID NO: 42) (SEQ ID NO: 750) 339
TGFBR2 ATTTGGAGCGAGGAATTT (SEQ ID NO: 43) (SEQ ID NO: 751) 340
TGFBR2 ATTTGGAGTGAGGAATTT (SEQ ID NO: 43) (SEQ ID NO: 752) 341
TGFBR2 TTGAAAGTCGGTTAAAGT (SEQ ID NO: 43) (SEQ ID NO: 753) 342
TGFBR2 TTGAAAGTTGGTTAAAGT (SEQ ID NO: 43) (SEQ ID NO: 754) 343
TGFBR2 AAAGTTTTCGGAGGGGTT (SEQ ID NO: 43) (SEQ ID NO: 907) 344
TGFBR2 AAAGTTTTTGGAGGGGTT (SEQ ID NO: 43) (SEQ ID NO: 908) 345
TGFBR2 GGTAGTTACGAGAGAGTT (SEQ ID NO: 43) (SEQ ID NO: 755) 346
TGFBR2 GGTAGTTATGAGAGAGTT (SEQ ID NO: 43) (SEQ ID NO: 756) 347
TIMP3 AGGTTTTTCGTTGGAGAA (SEQ ID NO: 44) (SEQ ID NO: 757) 348 TIMP3
AGGTTTTTTGTTGGAGAA (SEQ ID NO: 44) (SEQ ID NO: 758) 349 TIMP3
GAAAATATCGGTATTTTG (SEQ ID NO: 44) (SEQ ID NO: 759) 350 TIMP3
GAAAATATTGGTATTTTG (SEQ ID NO: 44) (SEQ ID NO: 760) 351 TIMP3
GGGATAAGCGAATTTTTT (SEQ ID NO: 44) (SEQ ID NO: 761) 352 TIMP3
GGGATAAGTGAATTTTTT (SEQ ID NO: 44) (SEQ ID NO: 762) 353 TIMP3
TTTTATTACGTATGTTTT (SEQ ID NO: 44) (SEQ ID NO: 763) 354 TIMP3
TTTTATTATGTATGTTTT (SEQ ID NO: 44) (SEQ ID NO: 764) 355 TP53
AAGTTGAACGTTTAGGTA (SEQ ID NO: 45) (SEQ ID NO: 765) 356 TP53
AAGTTGAATGTTTAGGTA (SEQ ID NO: 45) (SEQ ID NO: 766) 357 TP53
TTTTGAGTCGGTTTAAAG (SEQ ID NO: 45) (SEQ ID NO: 767) 358 TP53
TTTTGAGTTGGTTTAAAG (SEQ ID NO: 45) (SEQ ID NO: 768) 359 TP53
TATTTATTCGGTGTTGGG (SEQ ID NO: 45) (SEQ ID NO: 769) 360 TP53
TATTTATTTGGTGTTGGG (SEQ ID NO: 45) (SEQ ID NO: 770) 361 TP53
TTGGATTTCGAAATATTG (SEQ ID NO: 45) (SEQ ID NO: 771) 362 TP53
TTGGATTTTGAAATATTG (SEQ ID NO: 45) (SEQ ID NO: 772) 363 TP73
TGATTTAGCGTAGGTTTG (SEQ ID NO: 46) (SEQ ID NO: 773) 364 TP73
TGATTTAGTGTAGGTTTG (SEQ ID NO: 46) (SEQ ID NO: 774) 365 TP73
TTAGAGTTCGAGTTTATA (SEQ ID NO: 46) (SEQ ID NO: 775) 366 TP73
TTAGAGTTTGAGTTTATA (SEQ ID NO: 46) (SEQ ID NO: 776) 367 TP73
AAGTTACGGGTTTTATTG (SEQ ID NO: 46) (SEQ ID NO: 915) 368 TP73
AAGTTATGGGTTTTATTG (SEQ ID NO: 46) (SEQ ID NO: 916) 369 TP73
GGAAGTTTCGATGGTTTA (SEQ ID NO: 46) (SEQ ID NO: 777) 370 TP73
GGAAGTTTTGATGGTTTA (SEQ ID NO: 46) (SEQ ID NO: 778) 371 VHL
TTTATAAGCGTGATGATT (SEQ ID NO: 47) (SEQ ID NO: 779) 372 VHL
TTTATAAGTGTGATGATT (SEQ ID NO: 47) (SEQ ID NO: 780) 373 VHL
GGTGTTTTCGTGTGAGAT (SEQ ID NO: 47) (SEQ ID NO: 781) 374 VHL
GGTGTTTTTGTGTGAGAT (SEQ ID NO: 47) (SEQ ID NO: 782) 375 VHL
TGTGAGATGCGTTATTTT (SEQ ID NO: 47) (SEQ ID NO: 783) 376 VHL
TGTGAGATGTGTTATTTT (SEQ ID NO: 47) (SEQ ID NO: 784) 377 VHL
TATATTGCGCGTTTGATA (SEQ ID NO: 47) (SEQ ID NO: 785) 378 VHL
TATATTGTGTGTTTGATA (SEQ ID NO: 47) (SEQ ID NO: 786) 379 CDKN1C
ATGAAGAACGGTTAAGGG (SEQ ID NO: 48) (SEQ ID NO: 787) 380 CDKN1C
ATGAAGAATGGTTAAGGG (SEQ ID NO: 48) (SEQ ID NO: 788) 381 CDKN1C
TTTTATTTCGAGTTAGGT (SEQ ID NO: 48) (SEQ ID NO: 789) 382 CDKN1C
TTTTATTTTGAGTTAGGT (SEQ ID NO: 48) (SEQ ID NO: 790) 383 CDKN1C
TTAAGTTACGGTTATTAG (SEQ ID NO: 48) (SEQ ID NO: 791) 384 CDKN1C
TTAAGTTATGGTTATTAG (SEQ ID NO: 48) (SEQ ID NO: 792) 385 CDKN1C
TTAGTGTTCGTTTGGAAT (SEQ ID NO: 48) (SEQ ID NO: 793) 386 CDKN1C
TTAGTGTTTGTTTGGAAT (SEQ ID NO: 48) (SEQ ID NO: 794) 387 CAV1
TTGGTATCGTTGAAGAAT (SEQ ID NO: 49) (SEQ ID NO: 795) 388 CAV1
TTGGTATTGTTGAAGAAT (SEQ ID NO: 49) (SEQ ID NO: 796) 389 CAV1
TAGATTCGGAGGTAGGTA (SEQ ID NO: 49) (SEQ ID NO: 911) 390 CAV1
TAGATTTGGAGGTAGGTA (SEQ ID NO: 49) (SEQ ID NO: 912) 391 CAV1
TGGGGGTTCGAAAAAGTG (SEQ ID NO: 49) (SEQ ID NO: 797) 392 CAV1
TGGGGGTTTGAAAAAGTG (SEQ ID NO: 49) (SEQ ID NO: 798) 393 CAV1
GAAGTGTTCGTTTTTGTT (SEQ ID NO: 49) (SEQ ID NO: 799) 394 CAV1
GAAGTGTTTGTTTTTGTT (SEQ ID NO: 49) (SEQ ID NO: 800) 395 CDH13
GAAGTGGTCGTTAGTTTT (SEQ ID NO: 50) (SEQ ID NO: 801) 396 CDH13
GAAGTGGTTGTTAGTTTTT (SEQ ID NO: 50) (SEQ ID NO: 802) 397 CDH13
TTGTTTAGCGTGATTTGT (SEQ ID NO: 50) (SEQ ID NO: 803) 398 CDH13
TTGTTTAGTGTGATTTGT (SEQ ID NO: 50) (SEQ ID NO: 804) 399 CDH13
AAGGAATTCGTTTTGTAA (SEQ ID NO: 50) (SEQ ID NO: 903) 400 CDH13
AAGGAATTTGTTTTGTAA (SEQ ID NO: 50) (SEQ ID NO: 904) 401 CDH13
AATGTTTTCGTGATGTTG (SEQ ID NO: 50) (SEQ ID NO: 895) 402 CDH13
AATGTTTTTGTGATGTTG (SEQ ID NO: 50) (SEQ ID NO: 896) 403 DRG1
GAGTAGGACGGTGTTAAG (SEQ ID NO: 51) (SEQ ID NO: 805) 404 DRG1
GAGTAGGATGGTGTTAAG (SEQ ID NO: 51) (SEQ ID NO: 806) 405 DRG1
AAATTTAACGTTGGGTAG (SEQ ID NO: 51) (SEQ ID NO: 807) 406 DRG1
AAATTTAATGTTGGGTAG (SEQ ID NO: 51) (SEQ ID NO: 808) 407 DRG1
GATAATGACGGTGTTAGT (SEQ ID NO: 51) (SEQ ID NO: 809) 408 DRG1
GATAATGATGGTGTTAGT (SEQ ID NO: 51) (SEQ ID NO: 810) 409 DRG1
TGGTTGTACGTTAGGAGT (SEQ ID NO: 51) (SEQ ID NO: 811) 410 DRG1
TGGTTGTATGTTAGGAGT (SEQ ID NO: 51) (SEQ ID NO: 812) 411 PTGS2
TTTATTTTCGTGGGTAAA (SEQ ID NO: 52) (SEQ ID NO: 913) 412 PTGS2
TTTATTTTTGTGGGTAAA (SEQ ID NO: 52) (SEQ ID NO: 914) 413 PTGS2
AGTTATTTCGTTATATGG (SEQ ID NO: 52) (SEQ ID NO: 1007) 414 PTGS2
AGTTATTTTGTTATATGG
(SEQ ID NO: 52) (SEQ ID NO: 1008) 415 PTGS2 ATTTAAGGCGATTAGTTT (SEQ
ID NO: 52) (SEQ ID NO: 813) 416 PTGS2 ATTTAAGGTGATTAGTTT (SEQ ID
NO: 52) (SEQ ID NO: 814) 417 PTGS2 ATATTTGGCGGAAATTTG (SEQ ID NO:
52) (SEQ ID NO: 1023) 418 PTGS2 ATATTTGGTGGAAATTTG (SEQ ID NO: 52)
(SEQ ID NO: 1024) 419 THBS1 GGAGAGTTAGCGAGGGTT (SEQ ID NO: 53) (SEQ
ID NO: 815) 420 THBS1 GGAGAGTTAGTGAGGGTT (SEQ ID NO: 53) (SEQ ID
NO: 816) 421 THBS1 TATTTTAACGAATGGTTT (SEQ ID NO: 53) (SEQ ID NO:
817) 422 THBS1 TATTTTAATGAATGGTTT (SEQ ID NO: 53) (SEQ ID NO: 818)
423 THBS1 TTATAAAACGGGTTTAGT (SEQ ID NO: 53) (SEQ ID NO: 819) 424
THBS1 TTATAAAATGGGTTTAGT (SEQ ID NO: 53) (SEQ ID NO: 820) 425 THBS1
AGGTATTTCGGGAGATTA (SEQ ID NO: 53) (SEQ ID NO: 821) 426 THBS1
AGGTATTTTGGGAGATTA (SEQ ID NO: 53) (SEQ ID NO: 822) 427 TPEF
ATTTGTTTCGATTAATTT (=TMEFF2; =HPP1) (SEQ ID NO: 979) (SEQ ID NO:
54) 428 TPEF ATTTGTTTTGATTAATTT (=TMEFF2; =HPP1) (SEQ ID NO: 980)
(SEQ ID NO: 54) 429 TPEF ATAGGTTACGGGTTGGAG (=TMEFF2; =HPP1) (SEQ
ID NO: 917) (SEQ ID NO: 54) 430 TPEF ATAGGTTACGGGTTGGAG (=TMEFF2;
=HPP1) (SEQ ID NO: 918) (SEQ ID NO: 54) 431 TPEF AATTTGCGAACGTTTGGG
(=TMEFF2; =HPP1) (SEQ ID NO: 899) (SEQ ID NO: 54) 432 TPEF
AATTTGTGAATGTTTGGG (=TMEFF2; =HPP1) (SEQ ID NO: 900) (SEQ ID NO:
54) 433 DNMT1 AGTGGGTTCGTTTAAGTT (SEQ ID NO: 55) (SEQ ID NO: 823)
434 DNMT1 AGTGGGTTTGTTTAAGTT (SEQ ID NO: 55) (SEQ ID NO: 824) 435
DNMT1 TTTTTTACGCGGAGTAGT (SEQ ID NO: 55) (SEQ ID NO: 825) 436 DNMT1
TTTTTTATGTGGAGTAGT (SEQ ID NO: 55) (SEQ ID NO: 826) 437 DNMT1
GAGAGAGGCGATATTTTG (SEQ ID NO: 55) (SEQ ID NO: 827) 438 DNMT1
GAGAGAGGTGATATTTTG (SEQ ID NO: 55) (SEQ ID NO: 828) 439 DNMT1
GTATTAAACGGAGAGAGG (SEQ ID NO: 55) (SEQ ID NO: 829) 440 DNMT1
GTATTAAATGGAGAGAGG (SEQ ID NO: 55) (SEQ ID NO: 830) 441 CEA
AAGTGTTCGCGGTTGTTT (SEQ ID NO: 56) (SEQ ID NO: 1003) 442 CEA
AAGTGTTTGTGGTTGTTT (SEQ ID NO: 56) (SEQ ID NO: 1004) 443 CEA
TTTTGAGTCGTAGTTTAG (SEQ ID NO: 56) (SEQ ID NO: 831) 444 CEA
TTTTGAGTTGTAGTTTAG (SEQ ID NO: 56) (SEQ ID NO: 832) 445 CEA
AATAGATACGGAGAGGGA (SEQ ID NO: 56) (SEQ ID NO: 833) 446 CEA
AATAGATATGGAGAGGGA (SEQ ID NO: 56) (SEQ ID NO: 834) 447 MB
AGAAGGTGCGTGAGAGGT (SEQ ID NO: 57) (SEQ ID NO: 835) 448 MB
AGAAGGTGTGTGAGAGGT (SEQ ID NO: 57) (SEQ ID NO: 836) 449 MB
GGGTTAGTCGGGGTATTT (SEQ ID NO: 57) (SEQ ID NO: 837) 450 MB
GGGTTAGTTGGGGTATTT (SEQ ID NO: 57) (SEQ ID NO: 838) 451 MB
GGGGATAGCGAGTTATTG (SEQ ID NO: 57) (SEQ ID NO: 839) 452 MB
GGGGATAGTGAGTTATTG (SEQ ID NO: 57) (SEQ ID NO: 840) 453 MB
TTAGATTGCGTTATGGGG (SEQ ID NO: 57) (SEQ ID NO: 841) 454 MB
TTAGATTGTGTTATGGGG (SEQ ID NO: 57) (SEQ ID NO: 842) 455 PCNA
TAAAGAGGCGGGGAGATT (SEQ ID NO: 58) (SEQ ID NO: 1013) 456 PCNA
TAAAGAGGTGGGGAGATT (SEQ ID NO: 58) (SEQ ID NO: 1014) 457 PCNA
TATGGATACGATTGGTTT (SEQ ID NO: 58) (SEQ ID NO: 843) 458 PCNA
TATGGATATGATTGGTTT (SEQ ID NO: 58) (SEQ ID NO: 844) 459 PCNA
GTATTAAACGGTTGTAGG (SEQ ID NO: 58) (SEQ ID NO: 845) 460 PCNA
GTATTAAATGGTTGTAGG (SEQ ID NO: 58) (SEQ ID NO: 846) 461 PCNA
TTTGAAGTCGAAATTAGT (SEQ ID NO: 58) (SEQ ID NO: 847) 462 PCNA
TTTGAAGTTGAAATTAGT (SEQ ID NO: 58) (SEQ ID NO: 848) 463 CDC2
TGGAATTTCGATGTAAAT (SEQ ID NO: 59) (SEQ ID NO: 849) 464 CDC2
TGGAATTTTGATGTAAAT (SEQ ID NO: 59) (SEQ ID NO: 850) 465 CDC2
TAGTAGGACGATATTTTT (SEQ ID NO: 59) (SEQ ID NO: 851) 466 CDC2
TAGTAGGATGATATTTTT (SEQ ID NO: 59) (SEQ ID NO: 852) 467 CDC2
TAGTTATTCGGGAAGGTT (SEQ ID NO: 59) (SEQ ID NO: 853) 468 CDC2
TAGTTATTTGGGAAGGTT (SEQ ID NO: 59) (SEQ ID NO: 854) 469 CDC2
AAATTGTTCGTATTTGGT (SEQ ID NO: 59) (SEQ ID NO: 855) 470 CDC2
AAATTGTTTGTATTTGGT (SEQ ID NO: 59) (SEQ ID NO: 856) 471 ESR1
AGATATATCGGAGTTTGG (SEQ ID NO: 60) (SEQ ID NO: 857) 472 ESR1
AGATATATTGGAGTTTGG (SEQ ID NO: 60) (SEQ ID NO: 858) 473 ESR1
GTTTGGTACGGGGTATAT (SEQ ID NO: 60) (SEQ ID NO: 859) 474 ESR1
GTTTGGTATGGGGTATAT (SEQ ID NO: 60) (SEQ ID NO: 860) 475 ESR1
TTTTAAATCGAGTTGTGT (SEQ ID NO: 60) (SEQ ID NO: 861) 476 ESR1
TTTTAAATTGAGTTGTGT (SEQ ID NO: 60) (SEQ ID NO: 862) 477 ESR1
TATGAGTTCGGGAGATTA (SEQ ID NO: 60) (SEQ ID NO: 863) 478 ESR1
TATGAGTTTGGGAGATTA (SEQ ID NO: 60) (SEQ ID NO: 864) 479 ESR1
TGGAGGTTCGGGAGTTTA (SEQ ID NO: 60) (SEQ ID NO: 969) 480 ESR1
TGGAGGTTTGGGAGTTTA (SEQ ID NO: 60) (SEQ ID NO: 970) 481 CASP8
GAATGAGTCGAGGAAGGT (SEQ ID NO: 61) (SEQ ID NO: 865) 482 CASP8
GAATGAGTTGAGGAAGGT (SEQ ID NO: 61) (SEQ ID NO: 866) 483 CASP8
TATTGAGACGTTAAGTAA (SEQ ID NO: 61) (SEQ ID NO: 867) 484 CASP8
TATTGAGATGTTAAGTAA (SEQ ID NO: 61) (SEQ ID NO: 868) 485 CASP8
TAAGGTTACGTAGTTAGT (SEQ ID NO: 61) (SEQ ID NO: 869) 486 CASP8
TAAGGTTATGTAGTTAGT (SEQ ID NO: 61) (SEQ ID NO: 870) 487 CASP8
GTTAATAGCGGGGATTTT (SEQ ID NO: 61) (SEQ ID NO: 871) 488 CASP8
GTTAATAGTGGGGATTTT (SEQ ID NO: 61) (SEQ ID NO: 872) 489 RASSF1
GTAGTTTTCGAGAATGTT (SEQ ID NO: 62) (SEQ ID NO: 873) 490 RASSF1
GTAGTTTTTGAGAATGTT (SEQ ID NO: 62) (SEQ ID NO: 874) 491 RASSF1
TAATTAGAACGTTTTTTG (SEQ ID NO: 62) (SEQ ID NO: 875) 492 RASSF1
TAATTAGAATGTTTTTTG (SEQ ID NO: 62) (SEQ ID NO: 876) 493 RASSF1
TAGTTTTCGCGTAGAATT (SEQ ID NO: 62) (SEQ ID NO: 877) 494 RASSF1
TAGTTTTTGTGTAGAATT (SEQ ID NO: 62) (SEQ ID NO: 878) 495 RASSF1
TTTGTAGCGGGTGGAGTA (SEQ ID NO: 62) (SEQ ID NO: 995)
496 RASSF1 TTTGTAGTGGGTGGAGTA (SEQ ID NO: 62) (SEQ ID NO: 996) 497
MSH4 TTGTTTCGGCGGTTTTTT (SEQ ID NO: 63) (SEQ ID NO: 879) 498 MSH4
TTGTTTTGGTGGTTTTTT (SEQ ID NO: 63) (SEQ ID NO: 880) 499 MSH4
TTTTGGTACGTTAGGAGT (SEQ ID NO: 63) (SEQ ID NO: 881) 500 MSH4
TTTTGGTATGTTAGGAGT (SEQ ID NO: 63) (SEQ ID NO: 882) 501 MSH4
TAAATTTTCGGTTAGTTT (SEQ ID NO: 63) (SEQ ID NO: 883) 502 MSH4
TAAATTTTTGGTTAGTTT (SEQ ID NO: 63) (SEQ ID NO: 884) 503 MSH4
TTAGAGGTCGGTAGTTTA (SEQ ID NO: 63) (SEQ ID NO: 885) 504 MSH4
TTAGAGGTTGGTAGTTTA (SEQ ID NO: 63) (SEQ ID NO: 886) 505 MSH5
ATGTTTATCGTTTTGAGT (SEQ ID NO: 64) (SEQ ID NO: 887) 506 MSH5
ATGTTTATTGTTTTGAGT (SEQ ID NO: 64) (SEQ ID NO: 888) 507 MSH5
ATAGTTGTCGAATGTATG (SEQ ID NO: 64) (SEQ ID NO: 889) 508 MSH5
ATAGTTGTTGAATGTATG (SEQ ID NO: 64) (SEQ ID NO: 890) 509 MSH5
TAGAAGTGCGAAGGGGTA (SEQ ID NO: 64) (SEQ ID NO: 891) 510 MSH5
TAGAAGTGTGAAGGGGTA (SEQ ID NO: 64) (SEQ ID NO: 892) 511 MSH5
ATGTAATTCGAATGTTTT (SEQ ID NO: 64) (SEQ ID NO: 893) 512 MSH5
ATGTAATTTGAATGTTTT (SEQ ID NO: 64) (SEQ ID NO: 894)
[0091] TABLE-US-00003 TABLE 3 Oligonucleotides used in
differentiation between colon adenomas or carcinoma tissue and
healthy colon tissue. No: Gene Oligo: 1 CDH13 AATGTTTTCGTGATGTTG
(SEQ ID NO:50) (SEQ ID NO:895) 2 CDH13 AATGTTTTTGTGATGTTG (SEQ ID
NO:50) (SEQ ID NO:896) 3 CD44 TTGTTTAGCGGATTTTAG (SEQ ID NO:20)
(SEQ ID NO:897) 4 CD44 TTGTTTAGTGGATTTTAG (SEQ ID NO:20) (SEQ ID
NO:898) 5 TPEF AATTTGCGAACGTTTGGG (=TMEFF2; =HPP1) (SEQ ID NO:899)
(SEQ ID NO:54) 6 TPEF AATTTGTGAATGTTTGGG (=TMEFF2; =HPP1) (SEQ ID
NO:900) (SEQ ID NO:54) 7 CSPG2 GGGTAACGTCGAATTTAG (SEQ ID NO:21)
(SEQ ID NO:901) 8 CSPG2 GGGTAATGTTGAATTTAG (SEQ ID NO:21) (SEQ ID
NO:902) 9 CDH13 AAGGAATTCGTTTTGTAA (SEQ ID NO:50) (SEQ ID NO:903)
10 CDH13 AAGGAATTTGTTTTGTAA (SEQ ID NO:50) (SEQ ID NO:904) 11 GSTP1
GGAGTTCGCGGGATTTTT (SEQ ID NO:25) (SEQ ID NO:905) 12 GSTP1
GGAGTTTGTGGGATTTTT (SEQ ID NO:25) (SEQ ID NO:906) 13 TGFBR2
AAAGTTTTCGGAGGGGTT (SEQ ID NO:43) (SEQ ID NO:907) 14 TGFBR2
AAAGTTTTTGGAGGGGTT (SEQ ID NO:43) (SEQ ID NO:908) 15 N33
TGTTATTTCGGAGGGTTT (SEQ ID NO:36) (SEQ ID NO:909) 16 N33
TGTTATTTTGGAGGGTTT (SEQ ID NO:36) (SEQ ID NO:910) 17 CAV1
TAGATTCGGAGGTAGGTA (SEQ ID NO:49) (SEQ ID NO:911) 18 CAV1
TAGATTGGAGGTAGGTA (SEQ ID NO:49) (SEQ ID NO:912) 19 PTGS2
TTTATTTTTGTGGGTAAA (SEQ ID NO:52) (SEQ ID NO:913) 20 PTGS2
TTTATTTTTGTGGGTAAA (SEQ ID NO:52) (SEQ ID NO:914) 21 TP73
AAGTTACGGGTTTTATTG (SEQ ID NO:46) (SEQ ID NO:915) 22 TP73
AAGTTATGGGTTTTATTG (SEQ ID NO:46) (SEQ ID NO:916) 23 TPEF
ATAGGTTACGGGTTGGAG (=TMEFF2; =HPP1) (SEQ ID NO:917) (SEQ ID NO:54)
24 TPEF ATAGGTTATGGGTTGGAG (=TMEFF2; =HPP1) (SEQ ID NO:918) (SEQ ID
NO:54) 25 CD44 GTGGGGTTCGGAGGTATA (SEQ ID NO:20) (SEQ ID NO:919) 26
CD44 GTGGGGTTTGGAGGTATA (SEQ ID NO:20) (SEQ ID NO:920) 27 EYA4
AAGTAAGTCGTTGTTGTT (SEQ ID NO:24) (SEQ ID NO:921) 28 EYA4
AAGTAAGTTGTTGTTGTT (SEQ ID NO:24) (SEQ ID NO:922) 29 EYA4
AGTGTATGCGTAGAAGGT (SEQ ID NO:24) (SEQ ID NO:923) 30 EYA4
AGTGTATGTGTAGAAGGT (SEQ ID NO:24) (SEQ ID NO:924) 31 GTBP/MSH6
TTTGTTGGCGGGAAATTT (SEQ ID NO:26) (SEQ ID NO:925) 32 GTBP/MSH6
TTTGTTGGTGGGAAATTT (SEQ ID NO:26) (SEQ ID NO:926) 33 EGR4
GGAGTTTTCGGTATATAT (SEQ ID NO:4) (SEQ ID NO:927) 34 EGR4
GGAGTTTTTGGTATATAT (SEQ ID NO:4) (SEQ ID NO:928) 35 CDH1
TAGTGGCGTCGGAATTGT (SEQ ID NO:15) (SEQ ID NO:929) 36 CDH1
TAGTGGTGTTGGAATTGT (SEQ ID NO:15) (SEQ ID NO:930) 37 EGFR
ATTTGGTTCGATTTGGAT (SEQ ID NO:23) (SEQ ID NO:931) 38 EGFR
ATTTGGTTTGATTTGGAT (SEQ ID NO:23) (SEQ ID NO:932) 39 LKB1
AGGGAGGTCGTTGGTATT (SEQ ID NO:30) (SEQ ID NO:933) 40 LKB1
AGGGAGGTTGTTGGTATT (SEQ ID NO:30) (SEQ ID NO:934) 41 DAPK1
TTGTTTTTCGGAAATTTG (SEQ ID NO:22) (SEQ ID NO:935) 42 DAPK1
TTGTTTTTTGGAAATTTG (SEQ ID NO:22) (SEQ ID NO:936) 43 IGF2
GATTAGGGCGGGAAATAT (SEQ ID NO:29) (SEQ ID NO:937) 44 IGF2
GATTAGGGTGGGAAATAT (SEQ ID NO:29) (SEQ ID NO:938) 45 HLA-F
TATTTGGGCGGGTGAGTG (SEQ ID NO:10) (SEQ ID NO:939) 46 HLA-F
TATTTGGGTGGGTGAGTG (SEQ ID NO:10) (SEQ ID NO:940) 47 IGF2
TTGTATGGTCGAGTTTAT (SEQ ID NO:29) (SEQ ID NO:941) 48 IGF2
TTGTATGGTTGAGTTTAT (SEQ ID NO:29) (SEQ ID NO:942) 49 N33
GTTTAGTTAGCGGGTTTT (SEQ ID NO:36) (SEQ ID NO:943) 50 N33
GTTTAGTTAGTGGGTTTT (SEQ ID NO:36) (SEQ ID NO:944) 51 CSPG2
AAAAATTCGCGAGTTTAG (SEQ ID NO:21) (SEQ ID NO:945) 52 CSPG2
AAAAATTTGTGAGTTTAG (SEQ ID NO:21) (SEQ ID NO:946) 53 PTEN
TGATGTGGCGGGATTTTT (SEQ ID NO:39) (SEQ ID NO:947) 54 PTEN
TGATGTGGTGGGATTTTT (SEQ ID NO:39) (SEQ ID NO:948) 55 MLH1
TTAGGTAGCGGGTAGTAG (SEQ ID NO:32) (SEQ ID NO:949) 56 MLH1
TTAGGTAGTGGGTAGTAG (SEQ ID NO:32) (SEQ ID NO:950) 57 GTBP/MSH6
GAGGAATTCGGGTTTTAG (SEQ ID NO:26) (SEQ ID NO:951) 58 GTBP/MSH6
GAGGAATTTGGGTTTTAG (SEQ ID NO:26) (SEQ ID NO:952) 59 CALCA
ATTAGGTTCGTGTTTTAG (SEQ ID NO:14) (SEQ ID NO:953) 60 CALCA
ATTAGGTTTGTGTTTTAG (SEQ ID NO:14) (SEQ ID NO:954) 61 DAPK1
GAAGGGAGCGTATTTTAT (SEQ ID NO:22) (SEQ ID NO:955) 62 DAPK1
GAAGGGAGTGTATTTTAT (SEQ ID NO:22) (SEQ ID NO:956) 63 PTEN
AGAGTTATCGTTTTGTTT (SEQ ID NO:39) (SEQ ID NO:957) 64 PTEN
AGAGTTATTGTTTTGTTT (SEQ ID NO:39) (SEQ ID NO:958) 65 WT1
TGTTATATCGGTTAGTTG (SEQ ID NO:9) (SEQ ID NO:959) 66 WT1
TGTTATATTGGTTAGTTG (SEQ ID NO:9) (SEQ ID NO:960) 67 EGFR
TTTGTATTCGGAGTTGGG (SEQ ID NO:23) (SEQ ID NO:961) 68 EGFR
TTTGTATTTGGAGTTGGG (SEQ ID NO:23) (SEQ ID NO:962) 69 CSPG2
AAGATTTTCGGTTAGTTT (SEQ ID NO:21) (SEQ ID NO:963) 70 CSPG2
AAGATTTTTGGTTAGTTT (SEQ ID NO:21) (SEQ ID NO:964) 71 LKB1
TTTAGGTTCGTAAGTTTA (SEQ ID NO:30) (SEQ ID NO:965) 72 LKB1
TTTAGGTTTGTAAGTTTA (SEQ ID NO:30) (SEQ ID NO:966) 73 WT1
TATATTGGCGAAGGTTAA (SEQ ID NO:9) (SEQ ID NO:967) 74 WT1
TATATTGGTGAAGGTTAA (SEQ ID NO:9) (SEQ ID NO:968) 75 ESR1
TGGAGGTTCGGGAGTTTA (SEQ ID NO:60) (SEQ ID NO:969) 76 ESR1
TGGAGGTTTGGGAGTTTA (SEQ ID NO:60) (SEQ ID NO:970) 77 APC
TTTAATCGTATAGTTTGT (SEQ ID NO:12) (SEQ ID NO:971) 78 APC
TTTAATTGTATAGTTTGT (SEQ ID NO:12) (SEQ ID NO:972) 79 IGF2
AGTTTGAACGATGTAAGA (SEQ ID NO:29) (SEQ ID NO:973) 80 IGF2
AGTTTGAATGATGTAAGA
(SEQ ID NO:29) (SEQ ID NO:974) 81 MYOD1 AATTAGGTCGGATAGGAG (SEQ ID
NO:8) (SEQ ID NO:975) 82 MYOD1 AATTAGGTTGGATAGGAG (SEQ ID NO:8)
(SEQ ID NO:976) 83 N33 TTGGTTCGGGAAAGGTAA (SEQ ID NO:36) (SEQ ID
NO:977) 84 N33 TTGGTTTGGGAAAGGTAA (SEQ ID NO:36) (SEQ ID NO:978) 85
TPEF ATTTGTTTCGATTAATTT (=TMEFF2; =HPP1) (SEQ ID NO:979) (SEQ ID
NO:54) 86 TPEF ATTTGTTTTGATTAATTT (=TMEFF2; =HPP1) (SEQ ID NO:980)
(SEQ ID NO:54) 87 CDKN2a AATAGTTACGGTCGGAGG (SEQ ID NO:18) (SEQ ID
NO:981) 88 CDKN2a AATAGTTATGGTTGGAGG (SEQ ID NO:18) (SEQ ID NO:982)
89 CDH1 AGGGTTATCGCGTTTATG (SEQ ID NO:15) (SEQ ID NO:983) 90 CDH1
AGGGTTATTGTGTTTATG (SEQ ID NO:15) (SEQ ID NO:984) 91 APC
TATTTTGGCGGGTTGTAT (SEQ ID NO:12) (SEQ ID NO:985) 92 APC
TATTTTGGTGGGTTGTAT (SEQ ID NO:12) (SEQ ID NO:986)
[0092] TABLE-US-00004 TABLE 4 Oligonucleotides used in
differentiation between colon carcinoma tissue and healthy colon
tissue. No: Gene Oligo: 1 CDH13 AATGTTTTCGTGATGTTG (SEQ ID NO:50)
(SEQ ID NO:895) 2 CDH13 AATGTTTTTGTGATGTTG (SEQ ID NO:50) (SEQ ID
NO:896) 3 TPEF AATTTGCGAACGTTTGGG (=TMEFF2; =HPP1) (SEQ ID NO:899)
(SEQ ID NO:54) 4 TPEF AATTTGTGAATGTTTGGG (=TMEFF2; =HPP1) (SEQ ID
NO:900) (SEQ ID NO:54) 5 CDH13 AAGGAATTCGTTTTGTAA (SEQ ID NO:50)
(SEQ ID NO:903) 6 CDH13 AAGGAATTCGTTTTGTAA (SEQ ID NO:50) (SEQ ID
NO:904) 7 CSPG2 GGGTAACGTCGAATTTAG (SEQ ID NO:21) (SEQ ID NO:901) 8
CSPG2 GGGTAATGTTGAATTTAG (SEQ ID NO:21) (SEQ ID NO:902) 9 CD44
TTGTTTAGCGGATTTTAG (SEQ ID NO:20) (SEQ ID NO:897) 10 CD44
TTGTTTAGTGGATTTTAG (SEQ ID NO:20) (SEQ ID NO:898) 11 EYA4
AGTGTATGCGTAGAAGGT (SEQ ID NO:24) (SEQ ID NO:923) 12 EYA4
AGTGTATGTGTAGAAGGT (SEQ ID NO:24) (SEQ ID NO:924) 13 APC
TTTAATCGTATAGTTTGT (SEQ ID NO:12) (SEQ ID NO:971) 14 APC
TTTAATTGTATAGTTTGT (SEQ ID NO:12) (SEQ ID NO:972) 15 PTGS2
TTTATTTTCGTGGGTAAA (SEQ ID NO:52) (SEQ ID NO:913) 16 PTGS2
TTTATTTTTGTGGGTAAA (SEQ ID NO:52) (SEQ ID NO:914) 17 EYA4
AAGTAAGTCGTTGTTGTT (SEQ ID NO:24) (SEQ ID NO:921) 18 EYA4
AAGTAAGTTGTTGTTGTT (SEQ ID NO:24) (SEQ ID NO:922) 19 PTEN
ATTTTGCGTTCGTATTTA (SEQ ID NO:39) (SEQ ID NO:987) 20 PTEN
ATTTTGTGTTTGTATTTA (SEQ ID NO:39) (SEQ ID NO:988) 21 GSTP1
GGAGTTCGCGGGATTTTT (SEQ ID NO:25) (SEQ ID NO:905) 22 GSTP1
GGAGTTTGTGGGATTTTT (SEQ ID NO:25) (SEQ ID NO:906) 23 CAV1
TAGATTCGGAGGTAGGTA (SEQ ID NO:49) (SEQ ID NO:911) 24 CAV1
TAGATTTGGAGGTAGGTA (SEQ ID NO:49) (SEQ ID NO:912) 25 EGFR
ATTTGGTTCGATTTGGAT (SEQ ID NO:23) (SEQ ID NO:931) 26 EGFR
ATTTGGTTTGATTTGGAT (SEQ ID NO:23) (SEQ ID NO:932) 27 N33
TGTTATTTCGGAGGGTTT (SEQ ID NO:36) (SEQ ID NO:909) 28 N33
TGTTATTTTGGAGGGTTT (SEQ ID NO:36) (SEQ ID NO:910) 29 IGF2
TTGTATGGTCGAGTTTAT (SEQ ID NO:29) (SEQ ID NO:941) 30 IGF2
TTGTATGGTTGAGTTTAT (SEQ ID NO:29) (SEQ ID NO:942) 31 HLA-F
AGTTGTTTCGTAGATATT (SEQ ID NO:10) (SEQ ID NO:989) 32 HLA-F
AGTTGTTTTGTAGATATT (SEQ ID NO:10) (SEQ ID NO:990) 33 TPEF
ATAGGTTACGGGTTGGAG (=TMEFF2; =HPP1) (SEQ ID NO:917) (SEQ ID NO:54)
34 TPEF ATAGGTTATGGGTTGGAG (=TMEFF2; =HPP1) (SEQ ID NO:918) (SEQ ID
NO:54) 35 TP73 AAGTTACGGGTTTTATTG (SEQ ID NO:46) (SEQ ID NO:915) 36
TP73 AAGTTATGGGTTTTATTG (SEQ ID NO:46) (SEQ ID NO:916)
[0093] TABLE-US-00005 TABLE 5 Oligonucleotides used in
differentiation between colon adenoma tissue and healthy colon
tissue. No: Gene Oligo: 1 CD44 TTGTTTAGCGGATTTTAG (SEQ ID NO:20)
(SEQ ID NO:897) 2 CD44 TTGTTTAGTGGATTTTAG (SEQ ID NO:20) (SEQ ID
NO:898) 3 HLA-F TATTTGGGCGGGTGAGTG (SEQ ID NO:10) (SEQ ID NO:939) 4
HLA-F TATTTGGGTGGGTGAGTG (SEQ ID NO:10) (SEQ ID NO:940) 5 TGFBR2
AAAGTTTTCGGAGGGGTT (SEQ ID NO:43) (SEQ ID NO:907) 6 TGFBR2
AAAGTTTTTGGAGGGGTT (SEQ ID NO:43) (SEQ ID NO:908) 7 GTBP/MSH6
GAGGAATTCGGGTTTTAG (SEQ ID NO:26) (SEQ ID NO:951) 8 GTBP/MSH6
GAGGAATTTGGGTTTTAG (SEQ ID NO:26) (SEQ ID NO:952) 9 GTBP/MSH6
TTTGTTGGCGGGAAATTT (SEQ ID NO:26) (SEQ ID NO:925) 10 GTBP/MSH6
TTTGTTGGTGGGAAATTT (SEQ ID NO:26) (SEQ ID NO:926) 11 LKB1
AGGGAGGTCGTTGGTATT (SEQ ID NO:30) (SEQ ID NO:933) 12 LKB1
AGGGAGGTTGTTGGTATT (SEQ ID NO:30) (SEQ ID NO:934) 13 CD44
GTGGGGTTCGGAGGTATA (SEQ ID NO:20) (SEQ ID NO:919) 14 CD44
GTGGGGTTTGGAGGTATA (SEQ ID NO:20) (SEQ ID NO:920) 15 N33
GTTTAGTTAGCGGGTTTT (SEQ ID NO:36) (SEQ ID NO:943) 16 N33
GTTTAGTTAGTGGGTTTT (SEQ ID NO:36) (SEQ ID NO:944) 17 CDH13
AATGTTTTCGTGATGTTG (SEQ ID NO:50) (SEQ ID NO:895) 18 CDH13
AATGTTTTTGTGATGTTG (SEQ ID NO:50) (SEQ ID NO:896) 19 TP73
AAGTTACGGGTTTTATTG (SEQ ID NO:46) (SEQ ID NO:915) 20 TP73
AAGTTATGGGTTTTATTG (SEQ ID NO:46) (SEQ ID NO:916) 21 PTEN
TGATGTGGCGGGATTTTT (SEQ ID NO:39) (SEQ ID NO:947) 22 PTEN
TGATGTGGTGGGATTTTT (SEQ ID NO:39) (SEQ ID NO:948) 23 N33
TGTTATTTCGGAGGGTTT (SEQ ID NO:36) (SEQ ID NO:909) 24 N33
TGTTATTTTGGAGGGTTT (SEQ ID NO:36) (SEQ ID NO:910) 25 TPEF
AATTTGCGAACGTTTGGG (=TMEFF2; =HPP1) (SEQ ID NO:899) (SEQ ID NO:54
26 TPEF AATTTGTGAATGTTTGGG (=TMEFF2; =HPP1) (SEQ ID NO:900) (SEQ ID
NO:54) 27 GSTP1 GGAGTTCGCGGGATTTTT (SEQ ID NO:25) (SEQ ID NO:905)
28 GSTP1 GGAGTTTGTGGGATTTTT (SEQ ID NO:25) (SEQ ID NO:906) 29 EGFR
TTTGTATTCGGAGTTGGG (SEQ ID NO:23) (SEQ ID NO:961) 30 EGFR
TTTGTATTTGGAGTTGGG (SEQ ID NO:23) (SEQ ID NO:962) 31 RARB
TAGTAGTTCGGGTAGGGT (SEQ ID NO:40) (SEQ ID NO:991) 32 RARB
TAGTAGTTTGGGTAGGGT (SEQ ID NO:40) (SEQ ID NO:992) 33 N33
ATTTAGTTCGGGGGAGGA (SEQ ID NO:36) (SEQ ID NO:993) 34 N33
ATTTAGTTTGGGGGAGGA (SEQ ID NO:36) (SEQ ID NO:994) 35 CAV1
TAGATTCGGAGGTAGGTA (SEQ ID NO:49) (SEQ ID NO:911) 36 CAV1
TAGATTTGGAGGTAGGTA (SEQ ID NO:49) (SEQ ID NO:912) 37 TPEF
ATAGGTTACGGGTTGGAG (=TMEFF2; =HPP1) (SEQ ID NO:917) (SEQ ID NO:54)
38 TPEF ATAGGTTATGGGTTGGAG (=TMEFF2; =HPP1) (SEQ ID NO:918) (SEQ ID
NO:54) 39 CDKN2a AATAGTTACGGTCGGAGG (SEQ ID NO:18) (SEQ ID NO:981)
40 CDKN2a AATAGTTATGGTTGGAGG (SEQ ID NO:18) (SEQ ID NO:982) 41 N33
TTGGTTCGGGAAAGGTAA (SEQ ID NO:36) (SEQ ID NO:977) 42 N33
TTGGTTTGGGAAAGGTAA (SEQ ID NO:36) (SEQ ID NO:978) 43 MLH1
TTAGGTAGCGGGTAGTAG (SEQ ID NO:32) (SEQ ID NO:949) 44 MLH1
TTAGGTAGTGGGTAGTAG (SEQ ID NO:32) (SEQ ID NO:950) 45 APC
TATTTTGGCGGGTTGTAT (SEQ ID NO:12) (SEQ ID NO:985) 46 APC
TATTTTGGTGGGTTGTAT (SEQ ID NO:12) (SEQ ID NO:986) 47 CSPG2
GGGTAACGTCGAATTTAG (SEQ ID NO:21) (SEQ ID NO:901) 48 CSPG2
GGGTAATGTTGAATTTAG (SEQ ID NO:21) (SEQ ID NO:902) 49 CDH1
TAGTGGCGTCGGAATTGT (SEQ ID NO:15) (SEQ ID NO:929) 50 CDH1
TAGTGGTGTTGGAATTGT (SEQ ID NO:15) (SEQ ID NO:930) 51 PTGS2
TTTATTTTCGTGGGTAAA (SEQ ID NO:52) (SEQ ID NO:913) 52 PTGS2
TTTATTTTTGTGGGTAAA (SEQ ID NO:52) (SEQ ID NO:914) 53 RASSF1
TTTGTAGCGGGTGGAGTA (SEQ ID NO:62) (SEQ ID NO:995) 54 RASSF1
TTTGTAGTGGGTGGAGTA (SEQ ID NO:62) (SEQ ID NO:996) 55 WT1
TATATTGGCGAAGGTTAA (SEQ ID NO:9) (SEQ ID NO:967) 56 WT1
TATATTGGTGAAGGTTAA (SEQ ID NO:9) (SEQ ID NO:968) 57 CDKN2a
GGAGTTTTCGGTTGATTG (SEQ ID NO:18) (SEQ ID NO:997) 58 CDKN2a
GGAGTTTTTGGTTGATTG (SEQ ID NO:18) (SEQ ID NO:998) 59 ESR1
TGGAGGTTCGGGAGTTTA (SEQ ID NO:60) (SEQ ID NO:969) 60 ESR1
TGGAGGTTTGGGAGTTTA (SEQ ID NO:60) (SEQ ID NO:970) 61 IGF2
GATTAGGGCGGGAAATAT (SEQ ID NO:29) (SEQ ID NO:937) 62 IGF2
GATTAGGGTGGGAAATAT (SEQ ID NO:29) (SEQ ID NO:938) 63 MYOD1
AATTAGGTCGGATAGGAG (SEQ ID NO:8) (SEQ ID NO:975) 64 MYOD1
AATTAGGTTGGATAGGAG (SEQ ID NO:8) (SEQ ID NO:976) 65 CDH13
AAGGAATTCGTTTTGTAA (SEQ ID NO:50) (SEQ ID NO:903) 66 CDH13
AAGGAATTTGTTTTGTAA (SEQ ID NO:50) (SEQ ID NO:904) 67 EGR4
GGAGTTTTCGGTATATAT (SEQ ID NO:4) (SEQ ID NO:927) 68 EGR4
GGAGTTTTTGGTATATAT (SEQ ID NO:4) (SEQ ID NO:928) 69 S100A2
TATGTATACGAGTATTGG (SEQ ID NO:42) (SEQ ID NO:999) 70 S100A2
TATGTATATGAGTATTGG (SEQ ID NO:42) (SEQ ID NO:1000) 71 DAPK1
TTGTTTTTCGGAAATTTG (SEQ ID NO:22) (SEQ ID NO:935) 72 DAPK1
TTGTTTTTTGGAAATTTG (SEQ ID NO:22) (SEQ ID NO:936) 73 MGMT
AGTAGGATCGGGATTTTT (SEQ ID NO:31) (SEQ ID NO:1001) 74 MGMT
AGTAGGATTGGGATTTTT (SEQ ID NO:31) (SEQ ID NO:1002) 75 EYA4
AAGTAAGTCGTTGTTGTT (SEQ ID NO:24) (SEQ ID NO:921) 76 EYA4
AAGTAAGTTGTTGTTGTT (SEQ ID NO:24) (SEQ ID NO:922) 77 CEA
AAGTGTTCGCGGTTGTTT (SEQ ID NO:56) (SEQ ID NO:1003) 78 CEA
AAGTGTTTGTGGTTGTTT (SEQ ID NO:56) (SEQ ID NO:1004) 79 WT1
TGTTATATCGGTTAGTTG (SEQ ID NO:9) (SEQ ID NO:959) 80 WT1
TGTTATATTGGTTAGTTG
(SEQ ID NO:9) (SEQ ID NO:960) 81 GPIb beta GGAGTTCGGTCGGGTTTT (SEQ
ID NO:7) (SEQ ID NO:1005) 82 GPIb beta GGAGTTTGGTTGGGTTTT (SEQ ID
NO:7) (SEQ ID NO:1006) 83 CALCA ATTAGGTTCGTGTTTTAG (SEQ ID NO:14)
(SEQ ID NO:953) 84 CALCA ATTAGGTTTGTGTTTTAG (SEQ ID NO:14) (SEQ ID
NO:954) 85 PTGS2 AGTTATTTCGTTATATGG (SEQ ID NO:52) (SEQ ID NO:1007)
86 PTGS2 AGTTATTTTGTTATATGG (SEQ ID NO:52) (SEQ ID NO:1008) 87
MYOD1 GTGTTAGTCGTTTAGGGT (SEQ ID NO:8) (SEQ ID NO:1009) 88 MYOD1
GTGTTAGTTGTTTAGGGT (SEQ ID NO:8) (SEQ ID NO:1010) 89 EYA4
AGTGTATGCGTAGAAGGT (SEQ ID NO:24) (SEQ ID NO:923) 90 EYA4
AGTGTATGTGTAGAAGGT (SEQ ID NO:24) (SEQ ID NO:924) 91 CSPG2
AAAAATTCGCGAGTTTAG (SEQ ID NO:21) (SEQ ID NO:945) 92 CSPG2
AAAAATTTGTGAGTTTAG (SEQ ID NO:21) (SEQ ID NO:946) 93 PGR
TTAAGTGTCGGATTTGTG (SEQ ID NO:38) (SEQ ID NO:1011) 94 PGR
TTAAGTGTTGGATTTGTG (SEQ ID NO:38) (SEQ ID NO:1012) 95 PCNA
TAAAGAGGCGGGGAGATT (SEQ ID NO:58) (SEQ ID NO:1013) 96 PCNA
TAAAGAGGTGGGGAGATT (SEQ ID NO:58) (SEQ ID NO:1014) 97 MSH3
AGTATTTTCGTTTGAGGA (SEQ ID NO:34) (SEQ ID NO:1015) 98 MSH3
AGTATTTTTGTTTGAGGA (SEQ ID NO:34) (SEQ ID NO:1016) 99 WT1
TAGTGAGACGAGGTTTTT (SEQ ID NO:9) (SEQ ID NO:1017) 100 WT1
TAGTGAGATGAGGTTTTT (SEQ ID NO:9) (SEQ ID NO:1018) 101 MYC
TTATAATGCGAGGGTTTG (SEQ ID NO:35) (SEQ ID NO:1019) 102 MYC
TTATAATGTGAGGGTTTG (SEQ ID NO:35) (SEQ ID NO:1020) 103 HIC-1
TTTTAGAGCGTTAGGGTT (SEQ ID NO:27) (SEQ ID NO:1021) 104 HIC-1
TTTTAGAGTGTTAGGGTT (SEQ ID NO:27) (SEQ ID NO:1022) 105 PTGS2
ATATTTGGCGGAAATTTG (SEQ ID NO:52) (SEQ ID NO:1023) 106 PTGS2
ATATTTGGTGGAAATTTG (SEQ ID NO:52) (SEQ ID NO:1024) 107 EGFR
ATTTGGTTCGATTTGGAT (SEQ ID NO:23) (SEQ ID NO:931) 108 EGER
ATTTGGTTTGATTTGGAT (SEQ ID NO:23) (SEQ ID NO:932) 109 LKB1
TTTAGGTTCGTAAGTTTA (SEQ ID NO:30) (SEQ ID NO:965) 110 LKB1
TTTAGGTTTGTAAGTTTA (SEQ ID NO:30) (SEQ ID NO:966) 111 IGF2
TTGTATGGTCGAGTTTAT (SEQ ID NO:29) (SEQ ID NO:941) 112 IGF2
TTGTATGGTTGAGTTTAT (SEQ ID NO:29) (SEQ ID NO:942) 113 PTEN
AGAGTTATCGTTTTGTTT (SEQ ID NO:39) (SEQ ID NO:957) 114 PTEN
AGAGTTATTGTTTTGTTT (SEQ ID NO:39) (SEQ ID NO:958) 115 BCL2
TTTTGTTACGGTGGTGGA (SEQ ID NO:13) (SEQ ID NO:1025) 116 BCL2
TTTTGTTATGGTGGTGGA (SEQ ID NO:13) (SEQ ID NO:1026) 117 AR
AGAGGTTGCGTTTTAGAG (SEQ ID NO:5) (SEQ ID NO:1027) 118 AR
AGAGGTTGTGTTTTAGAG (SEQ ID NO:5) (SEQ ID NO:1028) 119 CDH1
AGGGTTATCGCGTTTATG (SEQ ID NO:15) (SEQ ID NO:983) 120 CDH1
AGGGTTATTGTGTTTATG (SEQ ID NO:15) (SEQ ID NO:984)
[0094] TABLE-US-00006 TABLE 6 Oligonucleotides used in
differentiation between colon carcinoma tissue and colon adenoma
tissue. No: Gene Oligo: 1 CDKN2a GTTGTTTTCGGTTGGTGT (SEQ ID NO:18)
(SEQ ID NO:1029) 2 CDKN2a GTTGTTTTTGGTTGGTGT (SEQ ID NO:18) (SEQ ID
NO:1030) 3 GPIb beta GGAGTTCGGTCGGGTTTT (SEQ ID NO:7) (SEQ ID
NO:1005) 4 GPIb beta GGAGTTTGGTTGGGTTTT (SEQ ID NO:7) (SEQ ID
NO:1006)
[0095] TABLE-US-00007 TABLE 7 Crossreference table to relate
numbers used in figure labelling to ID numbers used throughout the
document Number in Figures Gene name Healthy vs Non-Healthy 50-D
CDH13 20-C CD44 54-C TPEF (=TMEFF2; =HPP1) 21-C CSPG2 50-C CDH13
25-B GSTP1 43-C TGFBR2 36-B N33 49-A CAV1 52-C PTGS2 46-A TP73 54-B
TPEF (=TMEFF2; =HPP1) 20-A CD44 24-D EYA4 24-B EYA4 26-B GTBP/MSH6
4-C EGR4 15-E CDH1 23-E EGFR 30-B LKB1 22-D DAPK1 29-D IGF2 10-A
HLA-F 29-C IGF2 36-C N33 21-D CSPG2 39-D PTEN 32-B MLH1 26-A
GTBP/MSH6 14-C CALCA 22-C DAPK1 39-C PTEN 9-D WT1 23-A EGFR 21-A
CSPG2 30-A LKB1 9-C WT1 60-E ESR1 12-A APC 29-A IGF2 8-D MYOD1 36-A
N33 54-A TPEF (=TMEFF2; =HPP1) 18-E CDKN2a 15-D CDH1 12-C APC
Healthy vs Carcinoma 50-D CDH13 54-C TPEF (=TMEFF2; =HPP1) 50-C
CDH13 21-C CSPG2 20-C CD44 24-B EYA4 12-A APC 52-C PTGS2 24-D EYA4
39-B PGR 25-B GSTP1 49-A CAV1 23-E EGFR 36-B N33 29-C IGF2 10-D
HLA-F 54-B TPEF (=TMEFF2; =HPP1) 46-A TP73 Healthy vs Adenoma 20-C
CD44 10-A HLA-F 43-C TGFBR2 26-A GTBP/MSH6 26-B GTBP/MSH6 30-B LKB1
20-A CD44 36-C N33 50-D CDH13 46-A TP73 39-D PTEN 36-B N33 54-C
TPEF (=TMEFF2; =HPP1) 25-B GSTP1 23-A EGFR 40-A RARB 36-D N33 49-A
CAV1 54-B TPEF (=TMEFF2; =HPP1) 18-E CDKN2a 36-A N33 32-B MLH1 12-C
APC 21-C CSPG2 15-E CDH1 52-C PTGS2 62-D RASSF1 9-C WT1 18-D CDKN2a
60-E ESR1 29-D IGF2 8-D MYOD1 50-C CDH13 4-C EGR4 42-C S100A2 22-D
DAPK1 31-E MGMT 24-D EYA4 56-A CEA 9-D WT1 7-E GPIb beta 14-C CALCA
52-D PTGS2 8-B MYOD1 24-B EYA4 21-D CSPG2 38-C PGR 58-A PCNA 34-D
MSH3 9-B WT1 35-B MYC 27-C HIC-1 52-B PTGS2 23-E EGFR 30-A LKB1
29-C IGF2 39-C PTEN 13-D BCL2 5-B AR 15-D CDH1 Carcinoma vs Adenoma
18-B CDKN2a 7-E GPIb beta
[0096]
Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20060246433A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20060246433A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
* * * * *
References