title: Application of a Euryarchaeota-Specific Helicase from Thermococcus kodakarensis for Noise Reduction in PCR.
abstract: DNA/RNA helicases, which are enzymes for eliminating hydrogen bonds between bases of DNA/DNA, DNA/RNA, and RNA/RNA using the energy of ATP hydrolysis, contribute to various biological activities. In the present study, the Euryarchaeota-specific helicase EshA (TK0566) from the hyperthermophilic archaeon Thermococcus kodakarensis (Tk-EshA) was obtained as a recombinant form, and its enzymatic properties were examined. Tk-EshA exhibited maximal ATPase activity in the presence of RNA at 80°C. Unwinding activity was evaluated with various double-stranded DNAs (forked, 5' overhung, 3' overhung, and blunt end) at 50°C. Tk-EshA unwound forked and 3' overhung DNAs. These activities were expected to unwind the structured template and to peel off misannealed primers when Tk-EshA was added to a PCR mixture. To examine the effect of Tk-EshA on PCR, various target DNAs were selected, and DNA synthesis was investigated. When 16S rRNA genes were used as a template, several misamplified products (noise DNAs) were detected in the absence of Tk-EshA. In contrast, noise DNAs were eliminated in the presence of Tk-EshA. Noise reduction by Tk-EshA was also confirmed when Taq DNA polymerase (a family A DNA polymerase, PolI type) and KOD DNA polymerase (a family B DNA polymerase, α type) were used for PCR. Misamplified bands were also eliminated during toxA gene amplification from Pseudomonas aeruginosa DNA, which possesses a high GC content (69%). Tk-EshA addition was more effective than increasing the annealing temperature to reduce misamplified DNAs during toxA amplification. Tk-EshA is a useful tool to reduce noise DNAs for accurate PCR.
  
  
  
  PCR is a technique that is useful for genetic diagnosis, genetic engineering, and detection of pathogenic microorganisms. However, troubles with nonspecific DNA amplification often occur from primer misannealing. In order to achieve a specific DNA amplification by eliminating noise DNAs derived from primer misannealing, a thermostable Euryarchaeota-specific helicase (Tk-EshA) was included in the PCR mixture. The addition of Tk-EshA has reduced noise DNAs in PCR.
authors.affiliation: Department of Bioscience, Graduate School of Science and Technology, Kwansei-Gakuin University, Hyogo, Japan.
authors.forename: Ayako
authors.initials: A
authors.lastname: Fujiwara
authors.affiliation: Department of Bioscience, Graduate School of Science and Technology, Kwansei-Gakuin University, Hyogo, Japan.
authors.forename: Katsuhiro
authors.initials: K
authors.lastname: Kawato
authors.affiliation: Department of Bioscience, Graduate School of Science and Technology, Kwansei-Gakuin University, Hyogo, Japan.
authors.forename: Saori
authors.initials: S
authors.lastname: Kato
authors.affiliation: Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kyoto, Japan.
authors.forename: Kiyoshi
authors.initials: K
authors.lastname: Yasukawa
authors.affiliation: Department of Bioscience, Graduate School of Science and Technology, Kwansei-Gakuin University, Hyogo, Japan.
authors.forename: Ryota
authors.initials: R
authors.lastname: Hidese
authors.affiliation: Department of Bioscience, Graduate School of Science and Technology, Kwansei-Gakuin University, Hyogo, Japan fujiwara-s@kwansei.ac.jp.|Research Center for Intelligent Bio-Materials, Graduate School of Science and Technology, Kwansei-Gakuin University, Hyogo, Japan.
authors.forename: Shinsuke
authors.initials: S
authors.lastname: Fujiwara
mesh_terms: D036002:ADP Ribose Transferases; D000255:Adenosine Triphosphate / Q000378:metabolism; D001427:Bacterial Toxins; D004247:DNA / Q000378:metabolism; D004265:DNA Helicases / Q000235:genetics / Q000378:metabolism*; D004275:DNA, Ribosomal / Q000737:chemistry / Q000235:genetics; D005098:Exotoxins; D006358:Hot Temperature; D016133:Polymerase Chain Reaction / Q000379:methods*; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D019712:Thermococcus / Q000201:enzymology* / Q000235:genetics; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A
chemical_list: D036002:ADP Ribose Transferases; D000255:Adenosine Triphosphate; D001427:Bacterial Toxins; D004247:DNA; D004265:DNA Helicases; D004275:DNA, Ribosomal; D005098:Exotoxins; D012336:RNA, Ribosomal, 16S; D011994:Recombinant Proteins; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A