IDENTIFICATION AND CHARACTERIZATION OF DIFFERENTIALLY METHYLATED REGIONS OF GENOMIC DNA BY METHYLATION-SENSITIVE ARBITRARILY PRIMED PCR

We have developed a simple and reproducible fingerprinting method for screening the genome for regions of DNA that have altered patterns of DNA methylation associated with oncogenic transformation, Restriction enzymes with different sensitivities to cytosine methylation in their recognition sites were used to digest genomic DNAs from primary tumors , cell lines, and normal tissues prior to arbitrarily primed PCR ampli fication, Fragments that showed differential methylation were cloned a nd sequenced after resolving the PCR products on high-resolution polya crylamide gels. The cloned fragments were then used as probes for Sout hern analysis to confirm differential methylation of these regions in colon tissues and cell lines. Forty-four DNA fragments associated with a total of five different regions of genomic DNA containing methylati on sites We detected in 10 matched sets of normal and tumor colon DNAs and 7 colon cancer cell lines, A novel CPG island was also isolated t hat was found to be frequently hypermethylated in bladder and colon tu mors, We have demonstrated that this technique is a rapid and efficien t method that can be used to screen for altered methylation patterns i n genomic DNA and to isolate specific sequences associated with these changes,