Development of bioluminescent pyrophosphate assay using pyruvate phosphate dikinase and its application to single-nucleotide polymorphism analysis.
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.
Arakawa
Hidetoshi
H
School of Pharmaceutical Sciences, Showa University, Tokyo 142-8555, Japan. arakawa@pharm.showa-u.ac.jp
Karasawa
Koji
K
Munakata
Emi
E
Obinata
Rie
R
Maeda
Masako
M
Suzuki
Shigeya
S
Kamahori
Masao
M
Kambara
Hideki
H
D004247:DNA / Q000737:chemistry / Q000378:metabolism; D003838:Deoxyadenine Nucleotides / Q000737:chemistry / Q000378:metabolism; D003845:Deoxycytosine Nucleotides / Q000737:chemistry / Q000378:metabolism; D003848:Deoxyguanine Nucleotides / Q000737:chemistry / Q000378:metabolism; D011756:Diphosphates / Q000378:metabolism*; D008156:Luciferases / Q000378:metabolism; D008163:Luminescent Measurements / Q000191:economics / Q000379:methods*; D008954:Models, Biological; D020641:Polymorphism, Single Nucleotide; D011772:Pyruvate, Orthophosphate Dikinase / Q000378:metabolism*; D015203:Reproducibility of Results; D013873:Thionucleotides / Q000737:chemistry / Q000378:metabolism; D013942:Thymine Nucleotides / Q000737:chemistry / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism; D018631:ras Proteins / Q000235:genetics / Q000378:metabolism
D003838:Deoxyadenine Nucleotides; D003845:Deoxycytosine Nucleotides; D003848:Deoxyguanine Nucleotides; D011756:Diphosphates; D013873:Thionucleotides; D013942:Thymine Nucleotides; D016159:Tumor Suppressor Protein p53; C024107:2'-deoxycytidine 5'-triphosphate; C065990:alpha-thiodeoxyadenosine triphosphate; C029603:deoxyguanosine triphosphate; D004247:DNA; D008156:Luciferases; D011772:Pyruvate, Orthophosphate Dikinase; D018631:ras Proteins; C026600:2'-deoxyadenosine triphosphate; C024157:thymidine 5'-triphosphate