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Methylation‐Sensitive Representational Difference Analysis and Its Application to Cancer Research
Author(s) -
KANEDA ATSUSHI,
TAKAI DAIYA,
KAMINISHI MICHIO,
OKOCHI ERIKO,
USHIJIMA TOSHIKAZU
Publication year - 2003
Publication title -
annals of the new york academy of sciences
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.712
H-Index - 248
eISSN - 1749-6632
pISSN - 0077-8923
DOI - 10.1111/j.1749-6632.2003.tb05968.x
Subject(s) - hpaii , amplicon , methylation , dna methylation , biology , illumina methylation assay , genomic dna , restriction enzyme , methylated dna immunoprecipitation , representational difference analysis , cpg site , gene , microbiology and biotechnology , genetics , genomic imprinting , polymerase chain reaction , gene expression
A bstract : 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, Hpa II, and PCR to prepare ‘ Hpa II‐amplicons,’ followed by RDA. An Hpa II‐amplicon prepared using betaine and reverse electrophoresis was enriched 3.6‐fold (compared with the Hpa II‐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, Sac II and Nar I, 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.