[go: up one dir, main page]

US20150275312A1 - Method and nucleic acids for the analysis of a colon cell proliferative disorder - Google Patents

Method and nucleic acids for the analysis of a colon cell proliferative disorder Download PDF

Info

Publication number
US20150275312A1
US20150275312A1 US14/553,566 US201414553566A US2015275312A1 US 20150275312 A1 US20150275312 A1 US 20150275312A1 US 201414553566 A US201414553566 A US 201414553566A US 2015275312 A1 US2015275312 A1 US 2015275312A1
Authority
US
United States
Prior art keywords
seq
colon
methylation
dna
cpg
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
US14/553,566
Inventor
Peter Adorjan
Matthias Burger
Sabine Maier
Inko Nimmrich
Evelyne Becker
Ralf Lesche
Tamas Rujan
Armin Schmitt
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Epigenomics AG
Original Assignee
Epigenomics AG
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Epigenomics AG filed Critical Epigenomics AG
Priority to US14/553,566 priority Critical patent/US20150275312A1/en
Publication of US20150275312A1 publication Critical patent/US20150275312A1/en
Assigned to EPIGENOMICS AG reassignment EPIGENOMICS AG ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: SCHMITT, ARMIN, ADORJAN, PETER, BURGER, MATTHIAS, RUJAN, TAMAS, BECKER, EVELYNE, MAIER, SABINE, LESCHE, RALF, NIMMRICH, INKO
Abandoned legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/154—Methylation markers
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00—Chemistry: analytical and immunological testing
    • Y10T436/14—Heterocyclic carbon compound [i.e., O, S, N, Se, Te, as only ring hetero atom]
    • Y10T436/142222—Hetero-O [e.g., ascorbic acid, etc.]
    • Y10T436/143333—Saccharide [e.g., DNA, etc.]

Definitions

  • aspects of the invention relate generally to cellular proliferative disorders (e.g., cancer) and genomic DNA methylation, and more particularly to modified and genomic sequences, to oligonucleotides and/or PNA-oligomers for detecting the cytosine methylation state of genomic DNA, as well as to methods for ascertaining genetic and/or epigenetic parameters of genes for use in the differentiation, diagnosis, prognosis, treatment and/or monitoring of cellular proliferative disorders (e.g., cancer) including colon cell proliferative disorders, or the predisposition to colon cell proliferative disorders.
  • cellular proliferative disorders e.g., cancer
  • genomic DNA methylation e.g., genomic DNA methylation
  • methods for ascertaining genetic and/or epigenetic parameters of genes for use in the differentiation, diagnosis, prognosis, treatment and/or monitoring of cellular proliferative disorders (e.g., cancer) including colon cell proliferative disorders, or the predisposition to colon cell pro
  • 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.
  • 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.
  • aberrant DNA methylation constitutes one of the most prominent alterations and inactivates many tumor suppressor genes such as pl4ARF, pl6INK4a, THBS1, M3NT2, and M3NT31 and DNA mismatch repair genes such as hMLH1.
  • CIMP CpG island methylator phenotype
  • 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.
  • MIN microsatellite instability
  • CIMP-colon cancers evolve along a more classic genetic instability pathway (CIN), with a high rate of p53 mutations and chromosomal changes.
  • 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.
  • MIN tumours have a better prognosis than do tumours with a CIN phenotype and respond better to adjuvant chemotherapy.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Matrix Assisted Laser Desorption Ionization Mass Spectrometry 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.
  • 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.
  • the ionisation process via the matrix is considerably less efficient.
  • the selection of the matrix plays an eminently important role.
  • 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.
  • 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, THBS
  • 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 pre-disposition 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.
  • the present invention makes available methods and nucleic acids that allow the differentiation between colon carcinoma, colon adenoma and normal colon tissue.
  • the method enables the analysis of cytosine methylations and single nucleotide polymorphisms.
  • 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.
  • the panel as described herein may be adapted for use in the analysis of many aspects of colon cell proliferative disorders.
  • the method comprises the following steps:
  • the genomic DNA sample 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.
  • 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.
  • 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.
  • 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.
  • bisulfite solution is used for the reaction, then an addition takes place at the non-methylated cytosine bases.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • the fragments obtained by means of the amplification can carry a directly or indirectly detectable label.
  • 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).
  • MALDI matrix assisted laser desorption/ionisation mass spectrometry
  • ESI electron spray mass spectrometry
  • 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.
  • 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.
  • the amplificates hybridise to oligonucleotides previously bonded to a solid phase.
  • the oligonucleo-tides 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.
  • the cytosine of the CpG dinucleotide or in the case of TpG, the thiamine, is the 5 th to 9 th nucleotide from the 5′-end of the 10-mer.
  • One oligonucleotide exists for each CpG or TpG dinucleotide.
  • the non-hybridised amplificates are removed.
  • the hybridised amplificates are detected.
  • labels attached to the amplificates are identifiable at each position of the solid phase at which an oligonucleotide sequence is located.
  • 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).
  • MALDI matrix assisted laser desorption/ionisation mass spectrometry
  • ESI electron spray mass spectrometry
  • the produced fragments may have a single positive or negative net charge for better detectability in the mass spectrometer.
  • the aforementioned method is preferably used for ascertaining genetic and/or epigenetic parameters of genomic DNA.
  • 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.
  • the nucleic acids according to the present invention can be used for the analysis of genetic and/or epigenetic parameters of genomic DNA.
  • 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.
  • the modified nucleic acid could heretofore not be connected with the ascertainment of disease relevant genetic and epigenetic parameters.
  • 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.
  • oligonucleotides according to the present invention in which the cytosine of the CpG dinucleotide is the 5 th -9 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 4th-6th nucleotide from the 5′-end of the 9-mer.
  • 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.
  • a set which contains at least one oligomer for each of the CpG dinucleotides, from SEQ ID NO: 519 to SEQ ID NO: 1030.
  • a set comprising SEQ ID NO: 895 to SEQ ID NO: 1030.
  • oligonucleotide is bound to a solid phase. It is further preferred that all the oligonucleotides of one set are bound to a solid phase.
  • 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).
  • 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).
  • 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.
  • the set comprises SEQ ID NO: 519 to SEQ ID NO: 1030.
  • 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.
  • SNPs single nucleotide polymorphisms
  • an arrangement of different oligonucleotides and/or PNA-oligomers 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.
  • nitrocellulose as well as plastics such as nylon which can exist in the form of pellets or also as resin matrices are suitable alternatives.
  • 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.
  • 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 U.S. Pat. No. 5,744,305 by means of solid-phase chemistry and photolabile protecting groups.
  • 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 U.S. Pat. No. 5,837,832.
  • kits 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.
  • a kit along the lines of the present invention can also contain only part of the aforementioned components.
  • 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.
  • 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.
  • a further embodiment of the invention is a method for the analysis of the methylation status of genomic DNA without the need for pretreatment.
  • the genomic DNA sample 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.
  • 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.
  • 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.
  • the restriction fragments are amplified. In a preferred embodiment this is carried out using a polymerase chain reaction.
  • 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.
  • 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.
  • 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.
  • “genetic parameters” are mutations and polymorphisms of genomic DNA and sequences further required for their regulation.
  • mutations are, in particular, insertions, deletions, point mutations, inversions and polymorphisms and, particularly preferred, SNPs (single nucleotide polymorphisms).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 bisulfate.
  • 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 bisulfate and are subject to a preferred embodiment of this invention.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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).
  • genomic DNA was isolated from the cell samples using the Wizzard kit from (Promega).
  • the isolated genomic DNA from the samples are treated using a bisulfate 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.
  • PCR primers used are described in Table 1. PCR conditions were as follows.
  • 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.
  • each multiplex PCR product was diluted in 10 ⁇ Ssarc buffer (10 ⁇ Ssarc:230 ml 20 ⁇ SSC, 180 ml sodium lauroyl sarcosinate solution 20%, dilute to 1000 ml with dH20).
  • the reaction mixture was then hybridised to the detection oligonucleotides as follows. Denaturation at 95° C., cooling down to 10° C., hybridisation at 42° C. overnight followed by washing with 10 ⁇ Ssarc and dH20 at 42° C.
  • 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.
  • 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.
  • the SVM 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a fragment of the bisulfate 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.
  • gene fragments were amplified by PCR performing a first denaturation step for 14 min at 96° C., followed by 30-45 cycles (step 2: 60 sec at 96° C., step 3: 45 sec at 52° C., step 4: 75 sec at 72° C.) and a subsequent final elongation of 10 min at 72° C.
  • step 2 60 sec at 96° C.
  • step 3 45 sec at 52° C.
  • step 4 75 sec at 72° C.
  • the presence of PCR products was analysed by agarose gel electrophoresis.
  • 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.
  • 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

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Immunology (AREA)
  • Zoology (AREA)
  • Analytical Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Pathology (AREA)
  • General Health & Medical Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biophysics (AREA)
  • Hospice & Palliative Care (AREA)
  • General Engineering & Computer Science (AREA)
  • Oncology (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Physics & Mathematics (AREA)
  • Animal Behavior & Ethology (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Public Health (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Veterinary Medicine (AREA)
  • General Chemical & Material Sciences (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
  • Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

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.

Description

    CROSS REFERENCE TO RELATED APPLICATIONS
  • This application is a continuation application of U.S. application Ser. No. 10/506,111, filed Sep. 16, 2005, now pending; which is a 35 USC §371 National Stage application of International Application No. PCT/EP03/02035, filed Feb. 27, 2003, now expired; which claims the benefit of priority to EP 02002551.4, filed Feb. 27, 2002, now expired. The disclosure of each of the prior applications is considered part of, and is incorporated by reference in, the disclosure of this application.
    • BACKGROUND OF THE INVENTION
  • 1. Field of the Invention
  • Aspects of the invention relate generally to cellular proliferative disorders (e.g., cancer) and genomic DNA methylation, and more particularly to modified and genomic sequences, to oligonucleotides and/or PNA-oligomers for detecting the cytosine methylation state of genomic DNA, as well as to methods for ascertaining genetic and/or epigenetic parameters of genes for use in the differentiation, diagnosis, prognosis, treatment and/or monitoring of cellular proliferative disorders (e.g., cancer) including colon cell proliferative disorders, or the predisposition to colon cell proliferative disorders.
  • 2. Background Information
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 pl4ARF, pl6INK4a, THBS1, M3NT2, and M3NT31 and DNA mismatch repair genes such as hMLH1.
  • 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.
  • 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.
  • The identification of markers for the differentiation of colon carcinoma as well as for early detection are main goals of current research.
  • 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.
  • 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.
  • 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.
  • 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 HI 9 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).
  • 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, Buffing 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • 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 pre-disposition 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.
  • 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.
  • Furthermore, the method enables the analysis of cytosine methylations and single nucleotide polymorphisms.
  • 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.
  • In a preferred embodiment, the method comprises the following steps:
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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).
  • 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 oligonucleo-tides 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 5th to 9th nucleotide from the 5′-end of the 10-mer. One oligonucleotide exists for each CpG or TpG dinucleotide.
  • In the fifth step of the method, the non-hybridised amplificates are removed.
  • 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.
  • 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.
  • The aforementioned method is preferably used for ascertaining genetic and/or epigenetic parameters of genomic DNA.
  • 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.
  • The nucleic acids according to the present invention can be used for the analysis of genetic and/or epigenetic parameters of genomic DNA.
  • 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.
  • The modified nucleic acid could heretofore not be connected with the ascertainment of disease relevant genetic and epigenetic parameters.
  • 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 5th-9th 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 4th-6th nucleotide from the 5′-end of the 9-mer.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 U.S. Pat. No. 5,744,305 by means of solid-phase chemistry and photolabile protecting groups.
  • 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 U.S. Pat. No. 5,837,832.
  • 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.
  • 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.
  • The methods according to the present invention are vised, 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.
  • 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.
  • In the third step the restriction fragments are amplified. In a preferred embodiment this is carried out using a polymerase chain reaction.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • In the following, the present invention will be explained in greater detail on the basis of the sequences and examples without being limited thereto.
  • 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.
  • 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, RARE, SFN, S100A2, TGFBR2, TIMP3, TP53, TP73, VHL, CDKN1C, CAV1, CDH13, DRG1, PTGS2, THBS1, TPEF (=TMEFF2; =HPP1), DNMT1, CEA, MB, PCNA, CDC2, ESR1, CASP8, RASSFI, 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.
  • 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.
  • 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.
  • 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 bisulfate.
  • 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 bisulfate and are subject to a preferred embodiment of this invention.
    • BRIEF DESCRIPTION OF THE DRAWINGS
  • 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).
  • 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).
  • 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.
  • 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
  • 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
  • In the first step the genomic DNA was isolated from the cell samples using the Wizzard kit from (Promega).
  • The isolated genomic DNA from the samples are treated using a bisulfate 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.
  • 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 μM dNTPs
    2 pmol each primer
    • 1 U Hot Star Taq (Qiagen)
  • Forty cycles were carried out as follows. Denaturation at 95° C. for 15 min, followed by annealing at 55° C. for 45 sec., primer elongation at 65° C. for 2 min. A final elongation at 65° C. was carried out for 10 min.
  • 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.
  • 5 μl volume of each multiplex PCR product was diluted in 10×Ssarc buffer (10×Ssarc:230 ml 20×SSC, 180 ml sodium lauroyl sarcosinate solution 20%, dilute to 1000 ml with dH20). The reaction mixture was then hybridised to the detection oligonucleotides as follows. Denaturation at 95° C., cooling down to 10° C., hybridisation at 42° C. overnight followed by washing with 10×Ssarc and dH20 at 42° C.
  • 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
  • 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 vised. 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.
  • For selected distinctions, we trained a learning algorithm (support vector machine, SVM). The SVM (as discussed by F. Model, P. Adoijan, 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)
  • 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)
  • 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)
  • 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)
  • 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
  • A fragment of the bisulfate 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.
  • Genomic DNA was isolated from sample using the DNA wizzard DNA isolation kit (Pro-mega). Each sample was digested using Apa I according to manufacturer's recommendations (New England Biolabs).
  • 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 μM 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° C., followed by 30-45 cycles (step 2: 60 sec at 96° C., step 3: 45 sec at 52° C., step 4: 75 sec at 72° C.) and a subsequent final elongation of 10 min at 72° C. The presence of PCR products was analysed by agarose gel electrophoresis.
  • 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.
    • Tables
  • 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)
    AGATTTGGGGAAGAAGTTGTA
    (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 GAGAGGGAGTAAAAGAAAATGGT 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 TTTGGGAATGGGTTGTAT 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 TTGTATTAGGTTGGAAGTGGT 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
    (= TMEFF2; = 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 MSHS TATTAGGAATAAAGTTGGGGAG 395
    (SEQ ID NO: 64) (SEQ ID NO: 517)
    AACCCTTCAAACAAAAATAAAA
    (SEQ ID NO: 518)
  • 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 CACNA1G 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 AAGAGAAACGTTGGAATA
    (p27 Kip1) (SEQ ID NO: 619)
    (SEQ ID NO: 17)
    134 CDKN1B AAGAGAAATGTTGGAATA
    (p27 Kip1) (SEQ ID NO: 620)
    (SEQ ID NO: 17)
    135 CDKN1B TTTGATTTCGAGGGGAGT
    (p27 Kip1) (SEQ ID NO: 621)
    (SEQ ID NO: 17)
    136 CDKN1B TTTGATTTTGAGGGGAGT
    (p27 Kip1) (SEQ ID NO: 622)
    (SEQ ID NO: 17)
    137 CDKN1B GTATTTGGCGGTTGGATT
    (p27 Kip1) (SEQ ID NO: 623)
    (SEQ ID NO: 17)
    138 CDKN1B GTATTTGGTGGTTGGATT
    (p27 Kip1) (SEQ ID NO: 624)
    (SEQ ID NO: 17)
    139 CDKN1B TATAATTTCGGGAAAGAA
    (p27 Kip1) (SEQ ID NO: 625)
    (SEQ ID NO: 17)
    140 CDKN1B TATAATTTTGGGAAAGAA
    (p27 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 ATATTTAGTGAGTAGTGT
    (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 GTGGGGTTCGGAGGTATA
    (SEQ ID NO: 20) (SEQ ID NO: 920)
    161 CD44 GGTAGTTTCGATTATTTA
    (SEQ ID NO: 20) (SEQ ID NO: 639)
    162 CD44 GGTAGTTTCGATTATTTA
    (SEQ ID NO: 20) (SEQ ID NO: 640)
    163 CD44 TTGTTTAGCGGATTTTAG
    (SEQ ID NO: 20) (SEQ ID NO: 897)
    164 CD44 TCGTTTAGTGGATTTTAG
    (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 ATGTGATTCGTTTGGGTA
    (SEQ ID NO: 21) (SEQ ID NO: 644)
    171 CSPG2 GGGTAACGTCGAATTCAG
    (SEQ ID NO: 21) (SEQ ID NO: 901)
    172 CSPG2 GGGTAATGTTGAATTTAG
    (SEQ ID NO: 21) (SEQ ID NO: 902)
    173 CSPG2 AAAAATTCGCGAGTCTAG
    (SEQ ID NO: 21) (SEQ ID NO: 945)
    174 CSPG2 AAAAATTTGTGAGTTCAG
    (SEQ ID NO: 21) (SEQ ID NO: 946)
    175 DAPK1 GTTGGAGTCGAGGTTCGA
    (SEQ ID NO: 22) (SEQ ID NO: 645)
    176 DAPK1 GTTGGAGTCGAGGTTCGA
    (SEQ ID NO: 22) (SEQ ID NO: 646)
    177 DAPK1 TTTTCTGTCGGATTGGTG
    (SEQ ID NO: 22) (SEQ ID NO: 647)
    178 DAPK1 TTTTTCGTTGGATTGGTG
    (SEQ ID NO: 22) (SEQ ID NO: 648)
    179 DAPK1 GAAGGGAGCGTATTTCAT
    (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 EGFR TTTGTATTCGGAGTTGGG
    (SEQ ID NO: 23) (SEQ ID NO: 961)
    184 EGFR TTTGTATTTGGAGTTGGG
    (SEQ ID NO: 23) (SEQ ID NO: 962)
    185 EGFR GATGATTTCGAGGGTGTT
    (SEQ ID NO: 23) (SEQ ID NO: 649)
    186 EGFR GATGATTTTGAGGGTGTT
    (SEQ ID NO: 23) (SEQ ID NO: 650)
    187 EGFR GAGGGTTTCGTAGTGTTG
    (SEQ ID NO: 23) (SEQ ID NO: 651)
    188 EGFR GAGGGTTTTGTAGTGTTG
    (SEQ ID NO: 23) (SEQ ID NO: 652)
    189 EGFR TGGGGATTCGAATAAAGG
    (SEQ ID NO: 23) (SEQ ID NO: 653)
    190 EGFR TGGGGATTTGAATAAAGG
    (SEQ ID NO: 23) (SEQ ID NO: 654)
    191 EGFR ATTTGGTTCGATTTGGAT
    (SEQ ID NO: 23) (SEQ ID NO: 931)
    192 EGFR 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 GGTFTTITTGGTTAGTTG
    (SEQ ID NO: 25) (SEQ ID NO: 660)
    203 GSTP1 GGAGTTCGCGGGATTFTT
    (SEQ ID NO: 25) (SEQ ID NO: 905)
    204 GSTP1 GGAGTTTGTGGGATTTTT
    (SEQ ID NO: 25) (SEQ ID NO: 906)
    205 GSTP1 GTAGTFTTCGTTATTAGT
    (SEQ ID NO: 25) (SEQ ID NO: 661)
    206 GSTP1 GTAGTTFTTGITATTAGT
    (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 ITTUFTGGTGGGAAATIT
    (SEQ ID NO: 26) (SEQ ID NO: 926)
    211 GTBP/MSH6 TTTTGTCGGACGGAGTTT
    (SEQ ID NO: 26) (SEQ ID NO: 663)
    212 GTBP/MSH6 TTFTGTTGGATGGAGTFT
    (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 ATAGTTGTCGTTGAAGGG
    (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 TAAAAGGGCGTTTTGTGA
    (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 GATTTATTCGGAGAGGAG
    (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 TCTTTATGTGAGTTGTTT
    (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 TTAATTGCGGTTGTGTG
    (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 TTMP3 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 TTTTATTTCGAGTTAGGT
    (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 TTGGTATCGTTGAAGAAT
    (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 ATAGGTTATGGGTTGGAG
    (= 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 TTTGAGTTGTAGTTTAG
    (SEQ ID NO: 56) (SEQ ID NO: 832)
    445 CEA AA TAGATACGGAGAGGGA
    (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)
  • 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 TAGATTTGGAGGTAGGTA
    (SEQ ID NO: 49) (SEQ ID NO: 912)
    19 PTGS2 TTTATTTTCGTGGGTAAA
    (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 GAAGGGAGCGTATTTTAT
    (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)
  • 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 AAGGAATTTGTTTTGTAA
    (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)
  • 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 Al 1TAGTTCGGGGGAGGA
    (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 GGAGTTTGGTTGGGTHT
    (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 TAGTGAGATGAGGTHTT
    (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 EGFR ATTTGGTTCGATTTGGAT
    (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)
  • 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)
  • TABLE 7
    Crossreference table to relate numbers used in figure labelling to ID
    numbers used throughout the document
    Number in FIGS. 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

Claims (7)

What is claimed is:
1. A method for detecting colorectal cancer or colon adenomas or risk thereof in a human subject, comprising:
a) contacting genomic DNA in a biological sample obtained from the subject comprising colorectal DNA, with a reagent or series of reagents comprising at least one of bisulfite, hydrogen sulfite or disulfite;
b) comparing the nucleic acid sequence of untreated genomic DNA as set forth in SEQ ID NO: 54 to corresponding nucleic acid sequence derived from SEQ ID NO: 54 by bisulfite treatment according to method step a) based on the differential base-pairing properties of uracil and cytosine;
c) determining the methylation status of one or more CpG dinucleotides within the genomic region defined in method step b) by detecting unconverted cytosine bases in this region; and
d) detecting colorectal cancer or colon adenomas or risk thereof in the human subject by comparing the methylation state of at least one CpG position within the nucleic acid sequence of method step b) to the methylation state of the same CpG position or positions in genomic DNA derived from a human control subject not suffering from colorectal cancer or colon adenomas, wherein a higher degree of CpG methylation in the sample, compared to that of the control, indicates a presence or an increased likelihood of colorectal cancer or colon adenomas.
2. The method of claim 1, wherein in method step c) a portion or fragment of the treated DNA derived from SEQ ID NO: 54 is amplified by means of a polymerase chain reaction (PCR) to provide an amplificate.
3. The method of claim 2, wherein at least 10 different fragments, each fragment having a length of 10 to 200 base pairs, are amplified.
4. The method of claim 2, wherein the amplification step is carried out using one or more sets of primer oligonucleotides suitable to amplify a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 239, 240, 367 and 368 and amplifiable portions thereof.
5. The method of claim 2, wherein determining the methylation status of at least one CpG dinucleotide comprised by SEQ ID NO: 54 comprises hybridization of the amplificate to oligonucleotides or peptide nucleic acid (PNA) oligomers.
6. The method of claim 2, wherein the amplificates are labeled with a fluorescent label.
7. The method of claim 1, wherein the method differentiates between normal colon tissue and a tissue selected from the group consisting of colon adenoma tissue and colon carcinoma tissue.
US14/553,566 2002-02-27 2014-11-25 Method and nucleic acids for the analysis of a colon cell proliferative disorder Abandoned US20150275312A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US14/553,566 US20150275312A1 (en) 2002-02-27 2014-11-25 Method and nucleic acids for the analysis of a colon cell proliferative disorder

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
EP02004551.4 2002-02-27
EP02004551A EP1340818A1 (en) 2002-02-27 2002-02-27 Method and nucleic acids for the analysis of a colon cell proliferative disorder
US10/506,111 US20060246433A1 (en) 2002-02-27 2003-02-27 Method and nucleic acids for the analysis of a colon cell proliferative disorder
PCT/EP2003/002035 WO2003072821A2 (en) 2002-02-27 2003-02-27 Method and nucleic acids for the analysis of a colon cell proliferactive disorder
US14/553,566 US20150275312A1 (en) 2002-02-27 2014-11-25 Method and nucleic acids for the analysis of a colon cell proliferative disorder

Related Parent Applications (2)

Application Number Title Priority Date Filing Date
PCT/EP2003/002035 Continuation WO2003072821A2 (en) 2002-02-27 2003-02-27 Method and nucleic acids for the analysis of a colon cell proliferactive disorder
US10/506,111 Continuation US20060246433A1 (en) 2002-02-27 2003-02-27 Method and nucleic acids for the analysis of a colon cell proliferative disorder

Publications (1)

Publication Number Publication Date
US20150275312A1 true US20150275312A1 (en) 2015-10-01

Family

ID=27675653

Family Applications (6)

Application Number Title Priority Date Filing Date
US10/482,823 Expired - Fee Related US8691503B2 (en) 2002-02-27 2003-02-13 Method and nucleic acids for the analysis of colorectal cell proliferative disorders
US10/506,111 Abandoned US20060246433A1 (en) 2002-02-27 2003-02-27 Method and nucleic acids for the analysis of a colon cell proliferative disorder
US10/506,089 Abandoned US20060234224A1 (en) 2002-02-27 2003-02-27 Method and nucleic acids for the analysis of colon cell proliferative disorders
US12/794,449 Expired - Lifetime US9988683B2 (en) 2002-02-27 2010-06-04 Method and nucleic acids for the analysis of colon cell proliferative disorders
US14/186,984 Abandoned US20140179563A1 (en) 2002-02-27 2014-02-21 Method and Nucleic Acids for the Analysis of Colorectal Cell Proliferative Disorders
US14/553,566 Abandoned US20150275312A1 (en) 2002-02-27 2014-11-25 Method and nucleic acids for the analysis of a colon cell proliferative disorder

Family Applications Before (5)

Application Number Title Priority Date Filing Date
US10/482,823 Expired - Fee Related US8691503B2 (en) 2002-02-27 2003-02-13 Method and nucleic acids for the analysis of colorectal cell proliferative disorders
US10/506,111 Abandoned US20060246433A1 (en) 2002-02-27 2003-02-27 Method and nucleic acids for the analysis of a colon cell proliferative disorder
US10/506,089 Abandoned US20060234224A1 (en) 2002-02-27 2003-02-27 Method and nucleic acids for the analysis of colon cell proliferative disorders
US12/794,449 Expired - Lifetime US9988683B2 (en) 2002-02-27 2010-06-04 Method and nucleic acids for the analysis of colon cell proliferative disorders
US14/186,984 Abandoned US20140179563A1 (en) 2002-02-27 2014-02-21 Method and Nucleic Acids for the Analysis of Colorectal Cell Proliferative Disorders

Country Status (9)

Country Link
US (6) US8691503B2 (en)
EP (4) EP1340818A1 (en)
JP (3) JP2005518218A (en)
AT (3) ATE376596T1 (en)
AU (3) AU2003210265A1 (en)
CA (1) CA2451982A1 (en)
DE (3) DE60317045T2 (en)
ES (2) ES2295607T3 (en)
WO (3) WO2003072812A2 (en)

Families Citing this family (24)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9201067B2 (en) * 2003-03-05 2015-12-01 Posco Size-controlled macromolecule
EP1340818A1 (en) 2002-02-27 2003-09-03 Epigenomics AG Method and nucleic acids for the analysis of a colon cell proliferative disorder
US20040029128A1 (en) * 2002-08-08 2004-02-12 Epigenomics, Inc. Methods and nucleic acids for the analysis of CpG dinucleotide methylation status associated with the calcitonin gene
WO2005071111A1 (en) * 2004-01-23 2005-08-04 Lingvitae As Improving polynucleotide ligation reactions
US20090023134A1 (en) * 2004-03-17 2009-01-22 The Johns Hopkins University Neoplasia diagnostic compositions and methods of use
WO2006002344A1 (en) * 2004-06-23 2006-01-05 Epigenomics Ag Methods and nucleic acids for the detection of metastasis of colon cell proliferative disorders
KR100617649B1 (en) 2004-09-24 2006-09-04 (주)지노믹트리 Cancer diagnostic composition containing methylated promoter of colorectal cancer specific expression reducing gene and use thereof
ES2668911T3 (en) * 2004-09-30 2018-05-23 Epigenomics Ag Method for providing DNA fragments derived from an archived sample
WO2006088978A1 (en) 2005-02-16 2006-08-24 Epigenomics, Inc. Method for determining the methylation pattern of a polynucleic acid
US7932027B2 (en) 2005-02-16 2011-04-26 Epigenomics Ag Method for determining the methylation pattern of a polynucleic acid
ATE547537T1 (en) 2005-04-15 2012-03-15 Epigenomics Ag METHOD FOR DETERMINING DNA METHYLATION IN BLOOD OR URINE SAMPLES
US20100143899A1 (en) * 2005-05-17 2010-06-10 University Of Vermont And State Agricultural College Methods and products for diagnosing cancer
US20080076134A1 (en) * 2006-09-21 2008-03-27 Nuclea Biomarkers, Llc Gene and protein expression profiles associated with the therapeutic efficacy of irinotecan
US8445442B2 (en) * 2007-04-26 2013-05-21 University Of Vermont And State Agricultural College CCL18 and CCL3 methods and compositions for detecting and treating cancer
NZ584848A (en) * 2007-09-28 2012-09-28 Intrexon Corp Therapeutic gene-switch constructs and bioreactors for the expression of biotherapeutic molecules, and uses thereof
AU2008312457B2 (en) * 2007-10-19 2014-04-17 Genentech, Inc. Cysteine engineered anti-TENB2 antibodies and antibody drug conjugates
RU2565540C2 (en) * 2007-10-23 2015-10-20 Клиникал Джиномикс Пти. Лтд. Method of diagnosing neoplasm-ii
KR100969692B1 (en) * 2008-01-18 2010-07-14 한국생명공학연구원 Colorectal Cancer Diagnostic Markers Using Colorectal Cancer Overexpression Genes
US20100112713A1 (en) * 2008-11-05 2010-05-06 The Texas A&M University System Methods For Detecting Colorectal Diseases And Disorders
US20120053071A1 (en) * 2008-12-18 2012-03-01 Koninklijke Philips Electronics N.V. Method for the detection of dna methylation patterns
WO2012106525A2 (en) 2011-02-02 2012-08-09 Exact Sciences Corporation Digital sequence analysis of dna methylation
CA2852851A1 (en) * 2011-10-17 2013-04-25 King Abdullah University Of Science And Technology Composite biomarker for non-invasive screening, diagnosis and prognosis of colorectal cancer
US9598735B2 (en) * 2012-11-14 2017-03-21 JBS Science Inc. Detection of a panel of urine DNA markers for HCC screening and disease management
EP2886659A1 (en) * 2013-12-20 2015-06-24 AIT Austrian Institute of Technology GmbH Gene methylation based colorectal cancer diagnosis

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5487972A (en) * 1990-08-06 1996-01-30 Hoffmann-La Roche Inc. Nucleic acid detection by the 5'-3'exonuclease activity of polymerases acting on adjacently hybridized oligonucleotides

Family Cites Families (36)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5585481A (en) 1987-09-21 1996-12-17 Gen-Probe Incorporated Linking reagents for nucleotide probes
US5023090A (en) * 1989-08-16 1991-06-11 Levin Robert H Topical compositions containing LYCD and other topically active medicinal ingredients for the treatment of ACNE
US5457183A (en) 1989-03-06 1995-10-10 Board Of Regents, The University Of Texas System Hydroxylated texaphyrins
US5744101A (en) 1989-06-07 1998-04-28 Affymax Technologies N.V. Photolabile nucleoside protecting groups
US5580722A (en) * 1989-07-18 1996-12-03 Oncogene Science, Inc. Methods of determining chemicals that modulate transcriptionally expression of genes associated with cardiovascular disease
US5180808A (en) * 1989-11-27 1993-01-19 La Jolla Cancer Research Foundation Versican core protein, nucleic acid sequences encoding the same, nucleic acid probes, anti-versican antibodies, and methods of detecting the same
US5245022A (en) 1990-08-03 1993-09-14 Sterling Drug, Inc. Exonuclease resistant terminally substituted oligonucleotides
US5565552A (en) 1992-01-21 1996-10-15 Pharmacyclics, Inc. Method of expanded porphyrin-oligonucleotide conjugate synthesis
US5574142A (en) 1992-12-15 1996-11-12 Microprobe Corporation Peptide linkers for improved oligonucleotide delivery
SE501439C2 (en) 1993-06-22 1995-02-13 Pharmacia Lkb Biotech Method and apparatus for analyzing polynucleotide sequences
US5837832A (en) 1993-06-25 1998-11-17 Affymetrix, Inc. Arrays of nucleic acid probes on biological chips
EP1352956A1 (en) 1993-11-30 2003-10-15 McGILL UNIVERSITY Inhibition of DNA methyltransferase
US5597696A (en) 1994-07-18 1997-01-28 Becton Dickinson And Company Covalent cyanine dye oligonucleotide conjugates
US6017704A (en) * 1996-06-03 2000-01-25 The Johns Hopkins University School Of Medicine Method of detection of methylated nucleic acid using agents which modify unmethylated cytosine and distinguishing modified methylated and non-methylated nucleic acids
US5786146A (en) * 1996-06-03 1998-07-28 The Johns Hopkins University School Of Medicine Method of detection of methylated nucleic acid using agents which modify unmethylated cytosine and distinguishing modified methylated and non-methylated nucleic acids
AU6293798A (en) 1997-01-29 1998-08-18 Institut Pasteur Polynucleotide associated with the bo or bor syndrome, its corresponding polypeptide and diagnostic and therapeutic applications
DE19754482A1 (en) 1997-11-27 1999-07-01 Epigenomics Gmbh Process for making complex DNA methylation fingerprints
EP1084273A1 (en) * 1998-06-06 2001-03-21 Genostic Pharma Limited Probes used for genetic profiling
US7700324B1 (en) * 1998-11-03 2010-04-20 The Johns Hopkins University School Of Medicine Methylated CpG island amplification (MCA)
US5958773A (en) 1998-12-17 1999-09-28 Isis Pharmaceuticals Inc. Antisense modulation of AKT-1 expression
AU3316600A (en) 1999-02-22 2000-09-21 Torben F. Orntoft Gene expression in bladder tumors
US6331393B1 (en) * 1999-05-14 2001-12-18 University Of Southern California Process for high-throughput DNA methylation analysis
DE19935772C2 (en) * 1999-07-26 2002-11-07 Epigenomics Ag Method for the relative quantification of the methylation of cytosine bases in DNA samples
DK1232260T3 (en) * 1999-07-30 2008-02-04 Epidauros Biotechnologie Ag Polymorphism in the human MDR-1 gene and applications thereof
JP2004507214A (en) * 2000-03-15 2004-03-11 エピゲノミクス　アーゲー Diagnosis of tumor suppressor genes and diseases related to oncogenes
DE10019058A1 (en) * 2000-04-06 2001-12-20 Epigenomics Ag Designing primers and probes for analyzing diseases associated with cytosine methylation state e.g. arthritis, cancer, aging, arteriosclerosis comprising fragments of chemically modified genes associated with cell cycle
EP1268528A2 (en) * 2000-03-28 2003-01-02 Chiron Corporation Human genes and expression products
DE10029915B4 (en) * 2000-06-19 2005-06-16 Epigenomics Ag Method for the detection of cytosine methylations
WO2002000926A2 (en) * 2000-06-30 2002-01-03 Epigenomics Ag Diagnosis of diseases associated with signal transduction
MXPA03001834A (en) * 2000-09-01 2003-08-01 Epigenomics Ag Method for determining the degree of methylation of defined cytosines in genomic dna in the sequence context 5 -cpg-3.
US7029851B2 (en) * 2001-03-15 2006-04-18 Clemson University Polynucleotide encoding a gene conferring resistance to Bacillus thuringiensis toxins
DE10120798B4 (en) * 2001-04-27 2005-12-29 Genovoxx Gmbh Method for determining gene expression
CA2450379A1 (en) * 2001-06-10 2002-12-19 Irm Llc Molecular signatures of commonly fatal carcinomas
EP1470255A2 (en) * 2002-01-30 2004-10-27 Epigenomics AG Identification of cell differentiation states based on methylation patterns
EP1340818A1 (en) 2002-02-27 2003-09-03 Epigenomics AG Method and nucleic acids for the analysis of a colon cell proliferative disorder
WO2007123892A2 (en) * 2006-04-17 2007-11-01 Arqule Inc. Raf inhibitors and their uses

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5487972A (en) * 1990-08-06 1996-01-30 Hoffmann-La Roche Inc. Nucleic acid detection by the 5'-3'exonuclease activity of polymerases acting on adjacently hybridized oligonucleotides

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
Young et al. (PNAS January 2, 2001 Vol 98 p. 265-270) *

Also Published As

Publication number Publication date
ATE491038T1 (en) 2010-12-15
EP1478784A2 (en) 2004-11-24
WO2003072820A2 (en) 2003-09-04
EP1481095B1 (en) 2010-12-08
US20060234224A1 (en) 2006-10-19
JP2005518220A (en) 2005-06-23
AU2003210265A1 (en) 2003-09-09
EP1478778A2 (en) 2004-11-24
EP1481095A2 (en) 2004-12-01
EP1478778B1 (en) 2009-05-13
WO2003072821A2 (en) 2003-09-04
WO2003072821A3 (en) 2004-04-01
WO2003072812A2 (en) 2003-09-04
DE60317045D1 (en) 2007-12-06
AU2003248349B2 (en) 2008-05-01
US8691503B2 (en) 2014-04-08
DE60317045T2 (en) 2008-08-07
JP2005518219A (en) 2005-06-23
WO2003072820A3 (en) 2004-04-08
AU2003219002B2 (en) 2008-04-24
US9988683B2 (en) 2018-06-05
DE60327600D1 (en) 2009-06-25
US20060246433A1 (en) 2006-11-02
AU2003248349A1 (en) 2003-09-09
AU2003219002A1 (en) 2003-09-09
ATE376596T1 (en) 2007-11-15
ES2295607T3 (en) 2008-04-16
ES2325363T3 (en) 2009-09-02
US20110046005A1 (en) 2011-02-24
US20140179563A1 (en) 2014-06-26
EP1478784B1 (en) 2007-10-24
CA2451982A1 (en) 2003-09-04
US20050003463A1 (en) 2005-01-06
WO2003072812A3 (en) 2003-10-23
EP1340818A1 (en) 2003-09-03
DE60335245D1 (en) 2011-01-20
JP2005518218A (en) 2005-06-23
ATE431429T1 (en) 2009-05-15

Similar Documents

Publication Publication Date Title
US20150275312A1 (en) Method and nucleic acids for the analysis of a colon cell proliferative disorder
EP2115165B1 (en) Methods and nucleic acids for analyses of cell proliferative disorders
US20080145839A1 (en) Method and Nucleic Acids For the Differentiation of Astrocytoma, Oligoastrocytoma and Oligodenroglioma Tumor Cells
WO2004035806A2 (en) Methods and nucleic acids for the analysis of cpg dinucleotide methylation status associated with the calcitonin gene
WO2002077272A2 (en) Methods and nucleic acids for the analysis of hematopoietic cell proliferative disorders
EP3269826B1 (en) Methods and nucleic acids for the analysis of gene expression associated with the development of prostate cell proliferative disorders
US20070128592A1 (en) Method and nucleic acids for the analysis of a lung cell proliferative disorder
EP1360317A2 (en) Detection of aberrant dna methylation as marker for human cancer
US20060292564A1 (en) Method and nucleic acids for the analysis of breast cell proliferative disorders
US20040146868A1 (en) Methods and nucleic acids for the analysis of CpG dinucleotide methylation status associated with the development of peripheral zone prostate cancer
US20050064410A1 (en) Method and nucleic acids for the analysis of colon cancer
WO2005001142A2 (en) Methods and nucleic acids for the analysis of colorectal cell proliferative disorders
US9624530B2 (en) Methods for preservation of genomic DNA sequence complexity
EP1451354A2 (en) Method and nucleic acids for the analysis of a lymphoid cell proliferative disorder
US20040048278A1 (en) Diagnosis of diseases associated with the human c-mos gene
AU2001297759A1 (en) Detection of aberrant DNA methylation as marker for huiman cancer
AU2002333385A1 (en) Method and nucleic acids for the analysis of colon cancer
AU2002256709A1 (en) Methods and nucleic acids for the analysis of hematopoietic cell proliferative disorders

Legal Events

Date Code Title Description
AS Assignment

Owner name: EPIGENOMICS AG, GERMANY

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:BURGER, MATTHIAS;MAIER, SABINE;NIMMRICH, INKO;AND OTHERS;SIGNING DATES FROM 20050510 TO 20050707;REEL/FRAME:036806/0905

STCB Information on status: application discontinuation

Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION