Establishment of methylation-sensitive–representational difference analysis and isolation of hypo- and hypermethylated genomic fragments in mouse liver tumors

• DOI: 10.1073/pnas.94.6.2284
• Publication Date: 2002-07-26T14:43:20Z
• Authors: Toshikazu Ushijima; Keiichirou Morimura; Yoko Hosoya; Hideo Okonogi; Masae Tatematsu; Takashi Sugimura; Minako Nagao
• Keywords: Representational difference analysis; HpaII; Illumina Methylation Assay; CpG site; genomic DNA; Methylated DNA immunoprecipitation

Methylation of CpG sites in the genome, which is generally conserved during cell replication, considered to play important roles differentiation and carcinogenesis. However, investigations on changes methylation status have been limited known genes. To make a genome-wide search for differentially methylated genes, we developed methylation-sensitive–representational difference analysis (MS-RDA) method. The representation genome was prepared using methylation-sensitive restriction enzyme Hpa II, mixture ratio tester driver DNAs optimized detect differences single copy per diploid mammalian genome. By performing comparative MS-RDA one hepatocellular carcinoma background liver tissue mouse treated with food carcinogen (2-amino-3,4-dimethylimidazo[4,5- f ]quinoline), were able identify ( i ) extensive hypomethylation long interspersed nuclear element repetitive sequences number carcinomas, ii reduction gene dosage their mitochondrial DNA, iii hypermethylated DNA fragment unknown origin. Furthermore, by adding clones obtained first [MS-RDA w ith e limination xcessive c lones (MS-RDA-WEEC)], nine fragments that could not be detected at isolated as fragments. MS-RDA, combined MS-RDA-WEEC, thus promising approach two sources.