Cancer diagnostic method based upon DNA methylation differences

There is disclosed a cancer diagnostic method based upon DNA methylation differences at specific CpG sites. Specifically, the inventive method provides for a bisulfite treatment of DNA, followed by methylation-sensitive single nucleotide primer extension (Ms-SNuPE), for determination of strand-specific methylation status at cytosine residues.

TECHNICAL FIELD OF THE INVENTION
 The present invention provides a cancer diagnostic method based upon DNA
 methylation differences at specific CpG sites. Specifically, the inventive
 method provides for a bisulfite treatment of DNA, followed by
 methylation-sensitive single nucleotide primer extension (Ms-SNuPE), for
 determination of strand-specific methylation status at cytosine residues.
 BACKGROUND OF THE INVENTION
 Cancer treatments, in general, have a higher rate of success if the cancer
 is diagnosed early and treatment is started earlier in the disease
 process. The relationship between improved prognosis and stage of disease
 at diagnosis hold across all forms of cancer for the most part. Therefore,
 there is an important need to develop early assays of general
 tumorigenesis through marker assays that measure general tumorigenesis
 without regard to the tissue source or cell type that is the source of a
 primary tumor. Moreover, there is a need to address distinct genetic
 alteration patterns that can serve as a platform associated with general
 tumorigenesis for early detection and prognostic monitoring of many forms
 of cancer.
 Importance of DNA Methylation
 DNA methylation is a mechanism for changing the base sequence of DNA
 without altering its coding function. DNA methylation is a heritable,
 reversible and epigenetic change. Yet, DNA methylation has the potential
 to alter gene expression, which has profound developmental and genetic
 consequences. The methylation reaction involves flipping a target cytosine
 out of an intact double helix to allow the transfer of a methyl group from
 S-adenosylmethionine in a cleft of the enzyme DNA
 (cystosine-5)-methyltransferase (Klimasauskas et al., Cell 76:357-369,
 1994) to form 5-methylcytosine (5-mCyt). This enzymatic conversion is the
 only epigenetic modification of DNA known to exist in vertebrates and is
 essential for normal embryonic development (Bird, Cell 70:5-8, 1992; Laird
 and Jaenisch, Human Mol. Genet. 3:1487-1495, 1994; and Bestor and
 Jaenisch, Cell 69:915-926, 1992). The presence of 5-mCyt at CpG
 dinucleotides has resulted in a 5-fold depletion of this sequence in the
 genome during vertebrate evolution, presumably due to spontaneous
 deamination of 5-mCyt to T (Schoreret et al., Proc. Natl. Acad. Sci. USA
 89:957-961, 1992). Those areas of the genome that do not show such
 suppression are referred to as "CpG islands" (Bird, Nature 321:209-213,
 1986; and Gardiner-Garden et al., J. Mol. Biol. 196:261-282, 1987). These
 CpG island regions comprise about 1% of vertebrate genomes and also
 account for about 15% of the total number of CpG dinucleotides (Bird,
 Infra.). CpG islands are typically between 0.2 to about 1 kb in length and
 are located upstream of many housekeeping and tissue-specific genes, but
 may also extend into gene coding regions. Therefore, it is the methylation
 of cytosine residues within CpG islands in somatic tissues, which is
 believed to affect gene function by altering transcription (Cedar, Cell
 53:3-4, 1988).
 Methylation of cytosine residues contained within CpG islands of certain
 genes has been inversely correlated with gene activity. This could lead to
 decreased gene expression by a variety of mechanisms including, for
 example, disruption of local chromatin structure, inhibition of
 transcription factor-DNA binding, or by recruitment of proteins which
 interact specifically with methylated sequences indirectly preventing
 transcription factor binding. In other words, there are several theories
 as to how methylation affects mRNA transcription and gene expression, but
 the exact mechanism of action is not well understood. Some studies have
 demonstrated an inverse correlation between methylation of CpG islands and
 gene expression, however, most CpG islands on autosomal genes remain
 unmethylated in the germline and methylation of these islands is usually
 independent of gene expression. Tissue-specific genes are usually
 unmethylated and the receptive target organs but are methylated in the
 germline and in non-expressing adult tissues. CpG islands of
 constitutively-expressed housekeeping genes are normally unmethylated in
 the germline and in somatic tissues.
 Abnormal methylation of CpG islands associated with tumor suppressor genes
 may also cause decreased gene expression. Increased methylation of such
 regions may lead to progressive reduction of normal gene expression
 resulting in the selection of a population of cells having a selective
 growth advantage (i.e., a malignancy).
 It is considered that altered DNA methylation patterns, particularly
 methylation of cytosine residues, cause genome instability and are
 mutagenic. This, presumably, has led to an 80% suppression of a CpG methyl
 acceptor site in eukaryotic organisms, which methylate their genomes.
 Cytosine methylation further contributes to generation of polymorphism and
 germ-line mutations and to transition mutations that inactivate
 tumor-suppressor genes (Jones, Cancer Res. 56:2463-2467, 1996).
 Methylation is also required for embryonic development of mammals (Bestor
 and Jaenisch, Cell 69:915-926, 1992). It appears that that the methylation
 of CpG-rich promoter regions may be blocking transcriptional activity.
 Therefore, there is a probability that alterations of methylation are an
 important epigenetic criteria and can play a role in carcinogenesis in
 general due to its function of regulating gene expression. Ushijima et al.
 (Proc. Natl. Acad. Sci. USA 94:2284-2289, 1997) characterized and cloned
 DNA fragments that show methylation changes during murine
 hepatocarcinogenesis. Data from a group of studies of altered methylation
 sites in cancer cells show that it is not simply the overall levels of DNA
 methylation that are altered in cancer, but changes in the distribution of
 methyl groups.
 These studies suggest that methylation, at CpG-rich sequences known as CpG
 islands, provide an alternative pathway for the inactivation of tumor
 suppressors, despite the fact that the supporting studies have analyzed
 only a few restriction enzyme sites without much knowledge as to their
 relevance to gene control. These reports suggest that methylation of CpG
 oligonucleotides in the promoters of tumor suppressor genes can lead to
 their inactivation. Other studies provide data that suggest that
 alterations in the normal methylation process are associated with genomic
 instability (Lengauer et al. Proc. Natl. Acad. Sci. USA 94:2545-2550,
 1997). Such abnormal epigenetic changes may be found in many types of
 cancer and can, therefore, serve as potential markets for oncogenic
 transformation, provided that there is a reliable means for rapidly
 determining such epigenetic changes. The present invention was made to
 provide such a universal means for determining abnormal epigenetic changes
 and address this need in the art.
 Methods to Determine DNA Methylation
 There is a variety of genome scanning methods that have been used to
 identify altered methylation sites in cancer cells. For example, one
 method involves restriction landmark genomic scanning (Kawai et al., Mol.
 Cell. Biol. 14:7421-7427, 1994), and another example involves
 methylation-sensitive arbitrarily primed PCR (Gonzalgo et al., Cancer Res.
 57:594-599, 1997). Changes in methylation patterns at specific CpG sites
 have been monitored by digestion of genomic DNA with methylation-sensitive
 restriction enzymes followed by Southern analysis of the regions of
 interest (digestion-Southern method). The digestion-Southern method is a
 straightforward method but it has inherent disadvantages in that it
 requires a large amount of DNA (at least or greater than 5 .mu.g) and has
 a limited scope for analysis of CpG sites (as determined by the presence
 of recognition sites for methylation-sensitive restriction enzymes).
 Another method for analyzing changes in methylation patterns involves a
 PCR-based process that involves digestion of genomic DNA with
 methylation-sensitive restriction enzymes prior to PCR amplification
 (Singer-Sam et al., Nucl. Acids Res. 18:687,1990). However, this method
 has not been shown effective because of a high degree of false positive
 signals (methylation present) due to inefficient enzyme digestion of
 overamplification in a subsequent PCR reaction.
 Genomic sequencing has been simplified for analysis of DNA methylation
 patterns and 5-methylcytosine distribution by using bisulfite treatment
 (Frommer et al., Proc. Natl. Acad. Sci. USA 89:1827-1831, 1992). Bisulfite
 treatment of DNA distinguishes methylated from unmethylated cytosines, but
 original bisulfite genomic sequencing requires large-scale sequencing of
 multiple plasmid clones to determine overall methylation patterns, which
 prevents this technique from being commercially useful for determining
 methylation patterns in any type of a routine diagnostic assay.
 In addition, other techniques have been reported which utilize bisulfite
 treatment of DNA as a starting point for methylation analysis. These
 include methylation-specific PCR (MSP) (Herman et al. Proc. Natl. Acad.
 Sci. USA 93:9821-9826, 1992); and restriction enzyme digestion of PCR
 products amplified from bisulfite-converted DNA (Sadri and Hornsby, Nucl.
 Acids Res. 24:5058-5059, 1996; and Xiong and Laird, Nucl. Acids. Res.
 25:2532-2534, 1997).
 PCR techniques have been developed for detection of gene mutations
 (Kuppuswamy et al., Proc. Natl. Acad. Sci. USA 88:1143-1147, 1991) and
 quantitation of allelic-specific expression (Szabo and Mann, Genes Dev.
 9:3097-3108, 1995; and Singer-Sam et al., PCR Methods Appl. 1:160-163,
 1992). Such techniques use internal primers, which anneal to a
 PCR-generated template and terminate immediately 5' of the single
 nucleotide to be assayed. However an allelic-specific expression technique
 has not been tried within the context of assaying for DNA methylation
 patterns.
 Therefore, there is a need in the art to develop improved diagnostic assays
 for early detection of cancer using reliable and reproducible methods for
 determining DNA methylation patterns that can be performed using familiar
 procedures suitable for widespread use. This invention was made to address
 the foregoing need.
 SUMMARY OF THE INVENTION
 The present invention provides a method for determining DNA methylation
 patterns at cytosine sites, comprising the steps of:
 (a) obtaining genomic DNA from a DNA sample to be assayed;
 (b) reacting the genomic DNA with sodium bisulfite to convert unmethylated
 cytosine residues to uracil residues while leaving any 5-methylcytosine
 residues unchanged to provide primers specific for the bisulfite-converted
 genomic sample for top strand or bottom strand methylation analysis;
 (c) performing a PCR amplification procedure using the top strand or bottom
 strand specific primers;
 (d) isolating the PCR amplification products;
 (e) performing a primer extension reaction using Ms-SNuPE primers, [.sup.32
 P]dNTPs and Taq polymerase, wherein the Ms-SNuPE primers comprise from
 about a 15 mer to about a 22 mer length primer that terminates immediately
 5' of a single nucleotide to be assayed; and
 (f) determining the relative amount of methylation at CpG sites by
 measuring the incorporation of different .sup.32 P-labeled dNTPs.
 Preferably, the [.sup.32 P]NTP for top strand analysis is [.sup.32 P]dCTP
 or [.sup.32 P]TTP. Preferably, the [.sup.32 P]NTP for bottom strand
 analysis is [.sup.32 P]dATP or [.sup.32 P]dGTP. Preferably, the isolation
 step of the PCR products uses an electrophoresis technique. Most
 preferably, the electrophoresis technique uses an agarose gel. Preferably,
 the Ms-SNuPE primer sequence comprises a sequence of at least fifteen but
 no more than twenty five, bases having a sequence selected from the group
 consisting of GaL1 [SEQ ID NO. 1], GaL2 [SEQ ID NO. 2], GaL4 [SEQ ID NO.
 3], HuN1 [SEQ ID NO. 5], HuN2 [SEQ ID NO. 6], HuN3 [SEQ ID NO. 7], HuN4
 [SEQ ID NO. 8], HuN5 [SEQ ID NO. 8], HuN6 [SEQ ID NO. 9], CaS1 [SEQ ID NO.
 10], CaS2 [SEQ ID NO. 11], CaS4 [SEQ ID NO. 12], and combinations thereof.
 The present invention further provides a Ms-SNuPE primer sequence designed
 to anneal to and terminate immediately 5' of a desired cytosine codon in
 the CpG target site and that is located 5' upstream from a CpG island and
 are frequently hypermethylated in promoter regions of somatic genes in
 malignant tissue. Preferably, the Ms-SNuPE primer sequence comprises a
 sequence of at least fifteen bases having a sequence selected from the
 group consisting of GaL1 [SEQ ID NO. 1], GaL2 [SEQ ID NO. 2], GaL4 [SEQ ID
 NO. 3], HuN1 [SEQ ID NO. 5], HuN2 [SEQ ID NO. 6], HuN3 [SEQ ID NO. 7],
 HuN4 [SEQ ID NO. 8], HuN5 [SEQ ID NO. 8], HuN6 [SEQ ID NO. 9], CaS1 [SEQ
 ID NO. 10], CaS2 [SEQ ID NO. 11], CaS4 [SEQ ID NO. 12], and combinations
 thereof. The present invention further provides a method for obtaining a
 Ms-SNuPE primer sequence, comprising finding a hypermethylated CpG island
 in a somatic gene from a malignant tissue or cell culture, determining the
 sequence located immediately 5' upstream from the hypermethylated CpG
 island, and isolating a 15 to 25 mer sequence 5' upstream from the
 hypermethylated CpG island for use as a Ms-SNuPE primer. The present
 invention further provides a Ms-SNuPE primer comprising a 15 to 25 mer
 oligonucleotide sequence obtained by the process comprising, finding a
 hypermethylated CpG island in a somatic gene from a malignant tissue or
 cell culture, determining the sequence located immediately 5' upstream
 from the hypermethylated CpG island, and isolating a 15 to 25 mer sequence
 5' upstream from the hypermethylated CpG island for use as a Ms-SNuPE
 primer.

DETAILED DESCRIPTION OF THE INVENTION
 The present invention provides a method for determining DNA methylation
 patterns at cytosine sites, comprising the steps of:
 (a) obtaining genomic DNA from a DNA sample to be assayed, wherein sources
 of DNA include, for example, cell lines, blood, sputum, stool, urine,
 cerebrospinal fluid, paraffin-embedded tissues, histological slides and
 combinations thereof;
 (b) reacting the genomic DNA with sodium bisulfite to convert unmethylated
 cytosine residues to uracil residues while leaving any 5-methylcytosine
 residues unchanged to provide primers specific for the bisulfite-converted
 genomic sample for top strand or bottom strand methylation analysis;
 (c) performing a PCR amplification procedure using the top strand or bottom
 strand specific primers;
 (d) isolating the PCR amplification products;
 (e) performing a primer extension reaction using Ms-SNuPE primers, [.sup.32
 P]dNTPs and Taq polymerase, wherein the Ms-SNuPE primers comprise a from
 about a 15 mer to about a 22 mer length primer that terminates immediately
 5' of a single nucleotide to be assayed; and
 (f) determining the relative amount of allelic expression of CpG methylated
 sites by measuring the incorporation of different .sup.32 P-labeled dNTPs.
 Preferably, the [.sup.32 P]NTP for top strand analysis is [.sup.32 P]dCTP
 or [.sup.32 P]TTP. Preferably, the [.sup.32 P]NTP for bottom strand
 analysis is [.sup.32 P]dATP or [.sup.32 P]dGTP. Preferably, the isolation
 step of the PCR products uses an electrophoresis technique. Most
 preferably, the electrophoresis technique uses an agarose gel.
 DNA is isolated by standard techniques for isolating DNA from cellular,
 tissue or specimen samples. Such standard methods are found in textbook
 references such as Fritsch and Maniatis eds., Molecular Cloning: A
 Laboratory Manual, 1989.
 The bisulfite reaction is performed according to standard techniques. For
 example and briefly, approximately 1 microgram of genomic DNA (amount of
 DNA can be less when using micro-dissected DNA specimens) is denatured for
 15 minutes at 45.degree. C. with 2N NaOH followed by incubation with 0.1M
 hydroquinone and 3.6M sodium bisulfite (pH 5.0) at 55.degree. C. for 12
 hours (appropriate range is 4-12 hours). The DNA is then purified from the
 reaction mixture using standard (commercially-available) DNA miniprep
 columns, or other standard techniques for DNA purification are also
 appropriate. The purified DNA sample is resuspended in 55 microliters of
 water and 5 microliters of 3N NaOH is added for a desulfonation reaction,
 preferably performed at 40.degree. C. for 5-10 minutes. The DNA sample is
 then ethanol-precipitated and washed before being resuspended in an
 appropriate volume of water. Bisulfite treatment of DNA distinguishes
 methylated from unmethylated cytosines. The present bisulfite treatment
 method has advantages because it is quantitative, does not use restriction
 enzymes, and many CpG sites can be analyzed in each primer extension
 reaction by using a multiplex primer strategy.
 The PCR amplification step (c) can be performed by standard PCR techniques,
 following a manufacturer's instructions. For example, approximately 1-2
 microliters of the bisulfite-treated DNA was used as a template for
 strand-specific PCR amplification in a region of interest. In a PCR
 reaction profile for amplifying a portion of the p16 5' CpG island, for
 example, a procedure of initial denaturation of 94.degree. C. for 3
 minutes followed by a cycle of 94.degree. C. of 30 seconds, 68.degree. C.
 for 30 seconds, 72.degree. C. for 30 seconds for a total of 30 cycles. The
 PCR reactions were performed in 25 microliter volumes under conditions of:
 .about.50 ng bisulfite-converted DNA (less for micro dissected samples),
 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl.sub.2, 50 mM KCl, 0.1% gelatin/ml,
 100 .mu.M of each of dNTP, 0.5 .mu.M final concentration of each primer
 and 1 unit of Taq polymerase. There are many chromatographic techniques
 that can be used to isolate the PCR amplification products. In one
 illustrative procedure, approximately 10-25 microliters of the amplified
 PCR products were loaded onto 2% agarose gels and electrophoresed. The
 bands were visualized and isolated using standard get purification
 procedures.
 The primer extension reaction is conducted using standard PCR primer
 extension techniques but using Ms-SNuPE primers as provided herein.
 Approximately 10-50 nanograms of purified PCR template is used in each
 Ms-SNuPE reaction. A typical reaction volume is about 25 microliters and
 comprises PCR template (about 10-50 ng), 1.times.PCR buffer, 1 .mu.M of
 each Ms-SNuPE primer, 1 .mu.Ci of the appropriate .sup.32 P-labeled dNTP
 (either [.sup.32 P]dCTP, [.sup.32 P]TTP, [.sup.32 P]dATP, [.sup.32 P]dGTP
 or combinations thereof), and 1 unit of Taq polymerase. As a general rule,
 oligonucleotides used in the primer extension reactions were designed to
 have annealing temperatures within 2-3.degree. C. of each other and did
 not hybridize to sequences that originally contained CpG dinucleotides.
 The Ms-SNuPE reactions were performed at 95.degree. C. for 1 minute,
 50.degree. C. for 2 minutes, and 72.degree. C. for 1 minute. A stop
 solution (10 microliters) was added to the mixtures to terminate the
 reactions. The inventive Ms-SNuPE assay utilizes internal primer(s) which
 anneal to a PCR-generated template and terminate immediately 5' of the
 single nucleotide to be assayed. A similar procedure has been used
 successfully for detection of gene mutations Kuppuswamy et al., Proc.
 Natl. Acad. Sci. USA 88:1143-1147, 1991) and for quantitation of
 allele-specific expression (Szabo and Mann, Genes Dev. 9:3097-3108, 1995
 and Greenwood and Burke, Genome Res. 6:336-348, 1996).
 There are several techniques that are able to determine the relative amount
 of methylation at each CpG site, for example, using a denaturing
 polyacrylamide gel to measure .sup.32 P through phosphorimage analysis, or
 transfer of Ms-SNuPE reaction products to nylon membranes, or even using
 fluorescent probes instead of a .sup.32 P marker. In one method for
 determining the relative amount of methylation at each CpG site,
 approximately 1-2 microliters of each Ms-SNuPE reaction product was
 electrophoresed onto 15% denaturing polyacrylamide gel (7M urea). The gels
 were transferred to filter paper and then dried. Phosphorimage analysis
 was performed to determine the relative amount of radiolabeled
 incorporation. An alternative method for determining the relative amount
 of methylation at individual CpG sites is by a direct transfer of the
 Ms-SNuPE reaction products to nylon membranes. This technique can be used
 to quantitate an average percent methylation of multiple CpG sites without
 using polyacrylamide gel electrophoresis. High-throughput methylation
 analysis was performed by direct transfer of the Ms-SNuPE reactions onto
 nylon membranes. A total of 100 microliters or 0.4 mM NaOH, 1 mM Na.sub.4
 P.sub.2 O.sub.7 was added to the completed primer extension reactions
 instead of adding stop solution. The mixture was directly transferred to
 nylon membranes using a dotblot vacuum manifold in a 96 well plate format.
 Each vacuum transfer well was washed a total of 4 times with 200
 microliters of 2.times.SSC, 1 mM Na.sub.4 P.sub.2 O.sub.7. The entire
 membrane was washed in 2.times.SSC, 1 mM Na.sub.4 P.sub.2 O.sub.7. The
 radioactivity of each spot on the dried nylon membrane was quantitated by
 phosphorimaging analysis.
 In the inventive quantitative Ms-SNuPE assay, the relative amount of
 allelic expression is quantitated by measuring the incorporation of
 different .sup.32 P-labeled dNTPs. FIG. 1 outlines how the assay can be
 utilized for quantitative methylation analysis. For example, the initial
 treatment of genomic DNA with sodium bisulfite causes unmethylated
 cytosine to be converted to uracil, which is subsequently replicated as
 thymine during PCR. Methylcytosine is resistant to deamination and is
 replicated as cytosine during amplification. Quantitation of the ratio of
 methylated versus unmethylated cytosine (C versus T) at the original CpG
 sites can be determined by incubating a gel-isolated PCR product,
 primer(s) and Taq polymerase with either [.sup.32 P]dCTP or [.sup.32
 P]TTP, followed by denaturing polyacrylamide gel electrophoresis and
 phosphorimage analysis. In addition, opposite strand (bottom strand)
 Ms-SNuPE primers are further designed which would incorporate either
 [.sup.32 P]dATP or [.sup.32 P]dGTP to assess methylation status depending
 on which CpG site is analyzed.
 Ms-SNuPE Primers
 The present invention further provides a Ms-SNuPE primer sequence designed
 to anneal to and terminate immediately 5' of a desired cytosine codon in
 the CpG target site and that is located 5' upstream from a CpG island and
 are frequently hypermethylated in promoter regions of somatic genes in
 malignant tissue. Preferably, the Ms-SNuPE primer sequence comprises a
 sequence of at least fifteen bases having a sequence selected from the
 group consisting of GaL1 [SEQ ID NO. 1], GaL2 [SEQ ID NO. 2], GaL4 [SEQ ID
 NO. 3], HuN1 [SEQ ID NO. 5], HuN2 [SEQ ID NO. 6], HuN3 [SEQ ID NO. 7],
 HuN4 [SEQ ID NO. 8], HuN5 [SEQ ID NO. 8], HuN6 [SEQ ID NO. 9], CaS1 [SEQ
 ID NO. 10], CaS2 [SEQ ID NO. 11], CaS4 [SEQ ID NO. 12], and combinations
 thereof. The present invention further provides a method for obtaining a
 Ms-SNuPE primer sequence, comprising finding a hypermethylated CpG island
 in a somatic gene from a malignant tissue or cell culture, determining the
 sequence located immediately 5' upstream from the hypermethylated CpG
 island, and isolating a 15 to 25 mer sequence 5' upstream from the
 hypermethylated CpG island for use as a Ms-SNuPE primer. The present
 invention further provides a Ms-SNuPE primer comprising a 15 to 25 mer
 oligonucleotide sequence obtained by the process comprising, (a)
 identifying hypermethylated CpG islands a somatic gene from a malignant
 tissue or cell culture source, (b) determining the sequence located
 immediately 5' upstream from the hypermethylated CpG island, and (c)
 isolating at least a 15 mer sequence 5' upstream from the hypermethylated
 CpG island for use as a Ms-SNuPE primer. Preferably the Ms-SNuPE primer
 sequence is from about 15 to about 25 base pairs in length.
 The ability to detect methylation changes associated with oncogenic
 transformation is of critical importance in understanding how DNA
 methylation may contribute to tumorigenesis. Regions of DNA that have
 tumor-specific methylation alterations can be accomplished using a variety
 of techniques. This will permit rapid methylation analysis of specific CpG
 sites using the inventive quantitative Ms-SNuPE primer process. For
 example, techniques such as restriction landmark genomic scanning (RLGS)
 (Hatada et al., Proc. Natl. Acad. Sci. USA 88:9523-9527, 1995),
 methylation-sensitive-representational difference analysis (MS-RDA)
 (Ushijima et al., Proc. Natl. Acad. Sci. USA 94:2284-2289, 1997) and
 methylation-sensitive arbitrarily primed PCR (AP-PCR) (Gonzalgo et al.,
 Cancer Res. 57: 594-599, 1997) can be used for identifying and
 characterizing methylation differences between genomes.
 Briefly, sequence determinations of regions of DNA that show tumor-specific
 methylation changes can be performed using standard techniques, such as
 those procedures described in textbook references such as Fritsch and
 Maniatis eds., Molecular Cloning: A Laboratory Manual, 1989. Additionally,
 commercially available kits or automated DNA sequencing systems can be
 utilized. Once specific regions of DNA have been identified by using such
 techniques, the Ms-SNuPE primers can be applied for rapidly screening the
 most important CpG sites that are involved with the specific methylation
 changes associated with a cancer phenotype.
 EXAMPLE 1
 This example illustrates a quantitative methylation analysis of three top
 strand sites in a 5' CpG island of p16 in various DNA samples using the
 inventive method. The top panel provides the locations of three sites
 analyzed (numbered 1, 2 and 3) relative to the putative transcriptional
 start sites (vertical arrows pointing upwards) and the exon 1.alpha.
 coding domain. The PCR primers used for top strand amplification of the 5'
 region of p16 (which includes putative transcriptional start sites) were
 5'-GTA GGT GGG GAG GAG TTT AGT T-3' [SEQ ID NO. 13] and 5'-TCT AAT AAC
 CAACCA ACC CCT CC-3' [SEQ ID NO. 14]. The reactions were performed in 25
 .mu.l total volume under the conditions of 50 ng bisulfite-treated DNA, 10
 mM Tris-HCl (pH 8.3), 1.5 mM MgCl.sub.2, 50 mM KCl, 0.1% gelatin/ml, 100
 .mu.M of each dNTP, 0.5 .mu.M final concentration of each primer and 1 U
 of Taq polymerase (Boehinger Mannheim, Indianapolis, Ind.). The reactions
 were hot-started using a 1:1 mixture of Taq/TaqStart antibody (Clontech,
 Palo Alto, Calif.).
 An initial denaturation of 94.degree. C. for 3 minutes was followed by
 94.degree. C. for 30 sec. 68.degree. C. for 30 sec, 72.degree. C. for 30
 sec for a total of 35 cycles. The PCR products were separated by
 electrophoresis on 2% agarose gels and the bands were isolated using a
 Qiaquick.TM. gel extraction kit (Qiagen, Santa Clarita, Calif.).
 The Ms-SNuPE reaction was performed in a 25 ml reaction volume with 10-50
 ng of PCR template incubated in a final concentration of 1.times.PCR
 buffer, 1 .mu.M of each Ms-SNuPE primer, 1 .mu.Ci of either [.sup.32
 P]dCTP or [.sup.32 P]TTP and 1 U of Taq polymerase. The primer extensions
 were also hot-started using a 1: mixture of Taq/TaqStart antibody. The
 primers used for the Ms-SNuPE analysis were: site 1 5'-TTT TTT TGT TTG GAA
 AGA TAT-3' [SEQ ID NO. 15]; site 2 5'-TTT TAG GGG TGT TAT ATT-3' [SEQ ID
 NO. 16]; site 3 5-TTT GAG GGA TAG GGT-3' [SEQ ID NO. 17]. The conditions
 for the primer extension reactions were 95.degree. C. for 1 minute,
 50.degree. C. for 2 minutes and 70.degree. C. for 1 minute. A stop
 solution (10 .mu.l) was added to the reaction mixtures and the samples
 were loaded onto 15% denaturing polyacrylamide gels (7 M urea).
 Radioactivity of the bands was quantitated by phosphorimaging analysis.
 The control sets included "M" PCR product amplified from a plasmid
 containing bisulfide-specific methylated sequence; "U" PCR product
 amplified from a plasmid containing bisulfite-specific unmethylated
 sequence; and "mix" a 50:50 mixture of methylated and unmethylated
 PCR-amplified plasmid sequences. The DNA samples analyzed included T24 and
 J82 bladder cancer cell lines; wbc (white blood cell), melanoma (primary
 melanoma tumor tissue sample), and bladder (primary bladder tumor tissue
 sample). The tissue samples were micro dissected from paraffin-embedded
 tumor material. The grid at the bottom of the lower panel shows the ratio
 of methylated (C) versus unmethylated (T) bands at each site based upon
 phosphorimage quantitation.
 These data (FIG. 2) show the ability of the inventive assay to detect
 altered patterns of methylation.
 EXAMPLE 2
 This example illustrates a mixing experiment showing a linear response of
 the inventive Ms-SNuPE assay for detection of cytosine methylation. A T24
 bladder cancer cell line DNA (predominantly methylated) was added in
 increasing amounts to a J82 bladder cancer cell line DNA (predominantly
 unmethylated). FIG. 3 shows data from an 18 mer oligonucleotide [SEQ ID
 NO. 16] which was used in multiplex analysis of CpG methylation (site 2)
 of the p16 5'CpG in combination with a 15-mer and 21-mer primer [SEQ ID
 NOS 17 and 15, respectively] (correlation coefficient=0.99). Both the 15
 mer and 21-mer produced a nearly identical linear response as the 18-mer.
 FIG. 3 shows data from three separate experiments. Differential specific
 activity and incorporation efficiency of each [.sup.32 P]dNTP was
 controlled for by using a 50:50 mixture of bisulfite-specific methylated
 versus unmethylated PCR template for analysis.
 EXAMPLE 3
 This example provides a summary of DNA regions for which Ms-SNuPE primers
 can be designed and the inventive method applied for a quantitative
 detection of abnormal DNA methylation in cancer cells. The sequences are
 listed according to name, size and frequency of hypermethylation in the
 corresponding cell line or primary tumor.