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Establishment of Methylation-Sensitive-Representational Difference Analysis and Isolation of Hypo- and Hypermethylated Genomic Fragments in Mouse Liver Tumors
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Establishment of Methylation-Sensitive-Representational Difference Analysis and Isolation of Hypo- and Hypermethylated Genomic Fragments in Mouse Liver Tumors
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Establishment of Methylation-Sensitive-Representational Difference Analysis and Isolation of Hypo- and Hypermethylated Genomic Fragments in Mouse Liver Tumors
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Journal Article
Establishment of Methylation-Sensitive-Representational Difference Analysis and Isolation of Hypo- and Hypermethylated Genomic Fragments in Mouse Liver Tumors
1997
Overview
Methylation of CpG sites in the genome, which is generally conserved during cell replication, is considered to play important roles in cell differentiation and carcinogenesis. However, investigations on changes in methylation status have been limited to known genes. To make a genome-wide search for differentially methylated genes, we developed a methylation-sensitive-representational difference analysis (MS-RDA) method. The representation of the genome was prepared using the methylation-sensitive restriction enzyme HpaII, and the mixture ratio of tester and driver DNAs was optimized to detect differences in methylation status of a single copy per diploid mammalian genome. By performing comparative MS-RDA of one hepatocellular carcinoma and of background liver tissue of one mouse treated with a food carcinogen (2-amino-3,4-dimethylimidazo[4,5-f] quinoline), we were able to identify (i) extensive hypomethylation of long interspersed nuclear element repetitive sequences in a number of hepatocellular carcinomas, (ii) reduction of the gene dosage of their mitochondrial DNA, and (iii) a hypermethylated DNA fragment of unknown origin. Furthermore, by adding the clones obtained in the first MS-RDA to the driver DNA [MS-RDA with elimination of excessive clones (MS-RDA-WEEC)], nine DNA fragments that could not be detected at the first MS-RDA were isolated as differentially methylated DNA fragments. MS-RDA, combined with MS-RDA-WEEC, is thus a promising approach to identify DNA fragments differentially methylated in two DNA sources.
Publisher
National Academy of Sciences of the United States of America,National Acad Sciences,National Academy of Sciences,The National Academy of Sciences of the USA
