Methylation-sensitive representational difference analysis and its application to cancer research.
Methylation-sensitive representational difference analysis (MS-RDA) was previously established to detect differences in the methylation status of two genomes. This method uses the digestion of genomic DNA with a methylation-sensitive restriction enzyme, HpaII, and PCR to prepare "HpaII-amplicons," followed by RDA. An HpaII-amplicon prepared using betaine and reverse electrophoresis was enriched 3.6-fold (compared with the HpaII-amplicon prepared by the original method) with DNA fragments originating from CpG islands (CGIs). As for the specificity of MS-RDA, it was shown that DNA fragments that are unmethylated in the tester and almost completely methylated in the driver are efficiently isolated. This indicated that genes that are in biallelic methylation or in monoallelic methylation with loss of the other allele are efficiently isolated. Further, by use of two additional methylation-sensitive six-base recognition restriction enzymes, SacII and NarI, more DNA fragments were isolated from CGIs in the 5' regions of genes. After analysis of human lung, gastric, and breast cancers, 12 genes were seen to be silenced and additional genes seen to show decreased expression in association with methylation of genomic regions outside CGIs in the 5' regions of genes. MS-RDA is effective in identifying silenced genes in various cancers.
Carcinogenesis Division, National Cancer Center Research Institute, Tokyo 104-0045, Japan.
Atsushi
A
Kaneda
Daiya
D
Takai
Michio
M
Kaminishi
Eriko
E
Okochi
Toshikazu
T
Ushijima
D001483:Base Sequence; D019175:DNA Methylation; D004262:DNA Restriction Enzymes / Q000378:metabolism; D004273:DNA, Neoplasm / Q000737:chemistry / Q000235:genetics; D006801:Humans; D009369:Neoplasms / Q000235:genetics*
D004273:DNA, Neoplasm; D004262:DNA Restriction Enzymes