Analytical Sciences

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

Hidetoshi ARAKAWA*, Amane KOKADO*, Shuzo YOSHIZAWA*, Masako MAEDA*, Akifumi TOKITA** and Yuichiro YAMASHIRO**

*School of Pharmaceutical Sciences, Showa University, Shinagawa, Tokyo 142-8555, Japan
**Department of Pediatrics, Juntendo University School of Medicine, Bunkyo, Tokyo, 113-8431, Japan

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 reaction. 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. (Keywords: PCR, RFLP, ELISA, acetate kinase, firefly luciferase, bioluminescence, vitamin D receptor gene)

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