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

摘要

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.IMPORTANCE 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.