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Restriction landmark genome scanning method using isoschizomers ( Msp I/ Hpa II) for DNA methylation analysis
Author(s) -
Takamiya Tomoko,
Hosobuchi Saeko,
Asai Kenji,
Nakamura Eiji,
Tomioka Keisuke,
Kawase Makoto,
Kakutani Tetsuji,
Paterson Andrew H.,
Murakami Yasufumi,
Okuizumi Hisato
Publication year - 2006
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.200500776
Subject(s) - hpaii , isoschizomer , biology , dna methylation , genetics , methylation , restriction enzyme , illumina methylation assay , gene , ecotype , genomic dna , genome , restriction site , restriction fragment length polymorphism , microbiology and biotechnology , gene expression , polymerase chain reaction
Restriction landmark genome scanning (RLGS) is a 2‐DE of genomic DNA, which visualizes thousands of loci. In a conventional RLGS method for methylation analysis, we have used a methylation sensitive restriction enzyme, Not I as a landmark. However, it was unable to discriminate methylation polymorphism from sequence polymorphism. Here, we report an improved RLGS method to detect methylated sites directly. We employed isoschizomers, Msp I and Hpa II, that recognize the same sequence (CCGG) but have different methylation sensitivity. We carried out the RLGS analysis of Arabidopsis thaliana ecotype Columbia, and obtained a pair of spot patterns with Msp I and Hpa II. We detected 22 spots in both patterns. In comparison of them, 18% of the spots were polymorphic, which indicated the methylation of C 5m CGG sites. Further analyses revealed an additional methylated site of Not I. Moreover, 52 and 54 restriction enzyme sites were also analyzed in two other ecotypes, Wassilewskija and Landsberg erecta, respectively. Consequently, 15% of the 52 common sites showed methylation polymorphism among the three ecotypes. The restriction sites analyzed in this study were located in or near genes, and contribute new data about the correlation between methylation status and gene expression. Therefore, this result strongly indicates that the improved RLGS method is readily applicable to practical analyses of methylation dynamics, and provides clues to the relationship between methylation and gene expression.