Identification and Characterization of Differentially Methylated Regions of Genomic DNA by Methylation-sensitive Arbitrarily Primed PCR1

We have developeda simple and reproducible fingerprinting method for screeningthegenomefor regionsof DNA thathavealteredpatternsof DNA methylationassociatedwith oncogenictransformation.Restriction enzymes with different sensitivities to cytosine methylation in their rec ognition sites were used to digest genomic DNAs from primary tumors, cell lines, and normal tissuesprior to arbitrarily primed PCR amplifica tion.Fragmentsthatshowed ifferential methylationwereclonedand sequencedafter resolvingthe PCR productson high-resolutionpolyacryl amidegels.The clonedfragmentswerethenusedasprobesfor Southern analysisto confirm differential methylationof theseregionsin colon tissues and cell lines. Forty-four DNA fragments associated with a total of five differentregionsof genomicDNA containingmethylationsiteswere detectedin 10matchedsetsof normalandtumorcolonDNAsand7 colon cancercell lines.A novelCpG islandwasalsoisolatedthat wasfoundto be frequently hypermethylated in bladder and colon tumors. We have demonstratedthat thistechniqueisa rapidandefficientmethodthatcan beusedtoscreenfor alteredmethylationpatternsin genomicDNA andto isolatespecificsequences associatedwith thesechanges.