Application of a Euryarchaeota-Specific Helicase from Thermococcus kodakarensis for Noise Reduction in PCR

• Published in: Applied and environmental microbiology Vol. 82; no. 10; pp. 3022 - 3031
• Main Authors: Fujiwara, Ayako, Kawato, Katsuhiro, Kato, Saori, Yasukawa, Kiyoshi, Hidese, Ryota, Fujiwara, Shinsuke
• Format: Journal Article
• Language: English
• Published: United States American Society for Microbiology 15.05.2016
• Subjects: Adenosine Triphosphate - metabolism; ADP Ribose Transferases; Annealing; Bacterial Toxins; Biotechnology; Deoxyribonucleic acid; DNA; DNA - metabolism; DNA Helicases - genetics; DNA Helicases - metabolism; DNA, Ribosomal - chemistry; DNA, Ribosomal - genetics; Exotoxins; Genes; Hot Temperature; Microorganisms; Polymerase chain reaction; Polymerase Chain Reaction - methods; Pseudomonas aeruginosa; Pseudomonas aeruginosa Exotoxin A; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; RNA, Ribosomal, 16S - genetics; Thermococcus; Thermococcus - enzymology; Thermococcus - genetics; Virulence Factors
• ISSN: 0099-2240; 1098-5336
• DOI: 10.1128/aem.04116-15

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.