초고온 고세균 Thermococcus onnurineus NA1으로부터 dITPase의 특성 및 PCR에 적용분석

Abstract

In this study, we found that dITP could inhibit PCR amplification of various family B-type DNA polymerases, and 0.93% dITP was spontaneously generated from dATP during PCR amplification. Thus, it was hypothesized that the generated dITP might have negative effect on PCR amplification of family B-type DNA polymerases. To overcome the inhibitory effect of dITP during PCR amplification, a dITP pyrophosphatase (dITPase) from Thermococcus onnurineus NA1 (TNA1) was applied to PCR amplification. Genomic analysis of the hyperthermophilic archaeon T. onnurineus NA1 revealed the presence of a 555-bp open reading frame with 48% similarity to HAM1-like dITPase from Methanocaldococcus jannaschii DSM2661 (NP_247195). The dITPase-encoding gene was cloned and expressed in Escherichia coli. The purified protein hydrolyzed dITP, not dUTP. Addition of the purified protein to PCR reactions using DNA polymerases from T. onnurineus NA1 and Pyrococcus furiosus significantly increased product yield, overcoming the inhibitory effect of dITP. This study shows the first representation that removing dITP using a dITPase enhances the PCR amplification yield of family B-type DNA polymerase.[This work was supported by KORDI in-house program (PE98210) and the Marine and Extreme Genome Research Center program of Ministry of Land, Transport and Maritime Affairs, Republic of Korea]