Development of Bioluminescent DNA Analysis and Its Clinical Applications

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  • ARAKAWA Hidetoshi
    Department of Analytical Biochemistry, School of Pharmacy, Showa University
  • KARASAWA Koji
    Department of Analytical Biochemistry, School of Pharmacy, Showa University
  • SANO Yoshihiro
    Department of Analytical Biochemistry, School of Pharmacy, Showa University

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  • 生物発光DNA分析法の開発とその臨床への応用
  • セイブツ ハッコウ DNA ブンセキホウ ノ カイハツ ト ソノ リンショウ エ ノ オウヨウ

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Abstract

DNA analysis is an important technology with respect to the diagnosis of infectious disease and tailored medication. In this study, we developed a novel bioluminescent assay for pyrophosphate, which was applied to the detection of cariogenic bacteria in dental plaque, single nucleotide polymorphisms (SNPs) analysis and telomerase activity in cancer cells. We first developed a novel bioluminescent assay for the detection of pyrophosphate in a PCR product. The principle of this method is as follows: pyrophosphate released by PCR is converted to ATP by pyruvate phosphate dikinase (PPDK) in the presence of the substrate pyruvate phosphate and the coenzyme AMP; subsequently, the ATP concentration is determined by a firefly luciferase reaction. The detection limit of pyrophosphate is 1.56 × 10−15 mol/assay. This approach was applicable to allele-specific PCR products of Streptococcus mutans and Streptococcus sobrinus. In a following study, the bioluminescent assay for pyrophosphate was applied to SNPs analysis for the K-ras gene using a single-base extension reaction. The principle of this method is as follows: A specific primer within each aliquot possessing a short 3' end of the base of interest was hybridized to the single-stranded DNA template; subsequently, one of either α-S-dATP, dTTP, dGTP and dCTP, and (exo-) Klenow DNA polymerase were added and incubated for 1 min. Pyrophosphate released by DNA polymerase was converted to ATP by PPDK- the firefly luciferase reaction system described above. In third study, we developed a novel telomerase assay in cancer cells. In this assay, pyrophosphates generated by the telomerase reaction and PCR were detected by PPDK- the firefly luciferase reaction system described above. These methods, which do not require any expensive equipment, can be utilized to rapidly monitor cariogenic bacteria in dental plaque, one point mutation in p53 gene and the K-ras gene and tumor markers for clinical application.

Journal

  • BUNSEKI KAGAKU

    BUNSEKI KAGAKU 65 (9), 497-509, 2016

    The Japan Society for Analytical Chemistry

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