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Updated: Feb 10, 2026

Genotyping Single Nucleotide Polymorphisms in the Mitochondrial Genome by Pyrosequencing
Published on: February 10, 2023
Development of bioluminescent pyrophosphate assay using pyruvate phosphate dikinase and its application to
Hidetoshi Arakawa1, Koji Karasawa, Emi Munakata
1School of Pharmaceutical Sciences, Showa University, Tokyo 142-8555, Japan. arakawa@pharm.showa-u.ac.jp
Abstract:
DNA analysis is an important technology with respect to diagnosis of infectious disease and tailored medication. In this study, we developed a novel bioluminescent assay for pyrophosphate, and it was applied to single-nucleotide polymorphism (SNP) analysis using one-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, (exo-)Klenow DNA polymerase and one of either alpha-thio-dATP, dTTP, dGTP, or dCTP were added and incubated for 1 min. Pyrophosphate released by DNA polymerase is converted to ATP by pyruvate phosphate dikinase (PPDK), and the concentration of ATP is determined using the firefly luciferase reaction. This method, which does not require expensive equipment, can be used to rapidly monitor one point mutation in the gene.
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