Bioluminescent PCR-RFLP Enzyme-Linked Immunosorbent Assay for Analysis of Vitamin D Receptor Gene Polymorphism

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We developed a sensitive and rapid PCR-RFLP ELISA using acetate kinase (AK) and firefly luciferase as a detection system. AK used as a label enzyme could sensitively be detected by bioluminescent assay using the firefly luciferase reac-tion. The detection limit was 10-20 mol/assay and the luminescence was stable for 48 h. FITC-labeled sense primer and biotin labeled anti sense primer were used for PCR amplification of the vitamin D receptor gene. After PCR, the products were digested with Taq I or Apa I enzyme. The reaction products were diluted with assay buffer and transferred to a plate coated with anti FITC IgG. After incubation for 2 h at 37°C, the plate was washed and reacted with avidin/biotinylated AK, the AK activity was detected by bioluminescence assay using the firefly luciferin/luciferase system. DNA polymorphism types (AA, Aa, aa, TT, Tt, tt) of the vitamin D receptor gene (VDR) could be clearly determined by measuring the bioluminescent intensity or by using photon imaging with a CCD camera.

収録刊行物

  • Analytical sciences : the international journal of the Japan Society for Analytical Chemistry  

    Analytical sciences : the international journal of the Japan Society for Analytical Chemistry 15(10), 943-949, 1999-10-10 

    社団法人 日本分析化学会

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各種コード

  • NII論文ID(NAID)
    10004707766
  • NII書誌ID(NCID)
    AA10500785
  • 本文言語コード
    ENG
  • 資料種別
    ART
  • ISSN
    09106340
  • NDL 記事登録ID
    4872789
  • NDL 雑誌分類
    ZP4(科学技術--化学・化学工業--分析化学)
  • NDL 請求記号
    Z54-F482
  • データ提供元
    CJP書誌  CJP引用  NDL  J-STAGE 
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