Thermostable DNA helicase improves the sensitivity of digital PCR |
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Authors: | Ryota Hidese Katsuhiro Kawato Yukiko Nakura Ayako Fujiwara Kiyoshi Yasukawa Itaru Yanagihara Shinsuke Fujiwara |
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Affiliation: | 1. Department of Bioscience, Graduate School of Science and Technology, Kwansei-Gakuin University, 2-1 Gakuen, Sanda, Hyogo 669-1337, Japan;2. Department of Developmental Medicine, Research Institute, Osaka Women''s and Children''s Hospital, Osaka 594-1101, Japan;3. Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan |
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Abstract: | DNA/RNA helicases, which catalyze the unwinding of duplex nucleic acids using the energy of ATP hydrolysis, contribute to various biological functions involving DNA or RNA. Euryarchaeota-specific helicase Tk-EshA (superfamily 2) from the hyperthermophilic archaeon Thermococcus kodakarensis has been used to decrease generation of mis-amplified products (noise DNAs) during PCR. In this study, we focused on another type (superfamily 1B) of helicase, Tk-Upf1 (TK0178) from T. kodakarensis, and compared its effectiveness in PCR and digital PCR with that of Tk-EshA. For this purpose, we obtained Tk-Upf1 as a recombinant protein and assessed its enzymatic characteristics. Among various double-stranded DNA (dsDNA) substrates (forked, 5′ overhung, 3′ overhung, and blunt-ended duplex), Tk-Upf1 had the highest unwinding activity toward 5′ overhung DNAs. Noise DNAs were also eliminated in the presence of Tk-Upf1 at concentrations 10-fold lower than those required to yield a comparable reduction with Tk-EshA. When a 5′ or 3′ overhung mis-annealed primer was included as a competitive primer along with specific primers, noise DNAs derived from the mis-annealed primer were eliminated in the presence of Tk-Upf1. In digital PCR, addition of Tk-EshA or Tk-Upf1 increased fluorescent intensities and improved separation between common and risk allele clusters, indicating that both helicases functioned as signal enhancers. In comparison with Tk-EshA, a smaller amount of Tk-Upf1 was required to improve the performance of digital PCR. |
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Keywords: | Helicase Hyperthermophile PCR Noise reduction Digital PCR EshA Euryarchaeota-specific helicase SNPs single-nucleotide polymorphisms qPCR quantitative PCR CD circular dichroism CI 95% confidence interval dsDNA double-stranded DNA ssDNA single-stranded DNA |
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