A Novel Method, PCR-Mediated Amplification of Methylated Sites, Detects Methylated CpG Islands in the Genome of Bombyx mori

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An amplification-mediated method for the analysis of the methylation status was designed and applied to <i>Bombyx mori</i> DNA. The current method, named PAM, utilized the differential methylation sensitivities of four-base cutters, <i>Hap</i>II and <i>Msp</i>I. In order to reduce background signals, the first <i>Hap</i>II digestion step was followed by the ligation to the <i>Hap</i>II adaptor, while the second digestion by the same enzyme was followed by the Klenow-filling of the 5′ protruding ends. Then comes the step of digestion with either <i>Msp</i>I or <i>Hap</i>II, and further both digests were subjected to ligation with the <i>Msp</i>I adaptor and to PCR with primers annealing to the <i>Msp</i>I adaptor. The PCR products found in the <i>Msp</i>I digest, not in the <i>Hap</i>II digest, are expected to represent specifically the DNA sequences flanked by the two methylated CpGs in the context of CCGG. In fact, these types of specific signals were given by more than twenty fragments derived from DNA of a moth. By adding another primer that anneals to the <i>Hap</i>II adaptor at the PCR step allowed the detection of DNA fragments neighbored by a methylated site at either of the sides. The PAM method using genomic DNA of five larval tissues exhibited differences in the methylation pattern among them. These findings indicate that the PAM method is a powerful tool for screening genome-wide methylation status in the <i>B. mori</i> cells.

収録刊行物

  • Journal of insect biotechnology and sericology

    Journal of insect biotechnology and sericology 72(3), 191-195, 2003-10-31

    The Japanese Society of Sericultural Science

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各種コード

  • NII論文ID(NAID)
    10012453437
  • NII書誌ID(NCID)
    AA11558849
  • 本文言語コード
    ENG
  • 資料種別
    SHO
  • ISSN
    13468073
  • NDL 記事登録ID
    6730943
  • NDL 雑誌分類
    ZR7(科学技術--農林水産--農産)
  • NDL 請求記号
    Z54-J696
  • データ提供元
    CJP書誌  NDL  J-STAGE 
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