Most DNA polymerases cannot distinguish between thymine and

Most DNA polymerases cannot distinguish between thymine and uracil. However, family B DNA polymerases from Archaea, such as Pfu DNA polymerase, strongly bind to template strand uracil and stall polymerization in response to this TNP-470 [7], [8], [9]. The main inhibitor of PCR with Pfu DNA polymerase is dUTP generated by the deamination of dCTP at high temperatures [10]. Pyrococcus furiosus dUTPase is added to commercial preparations of thermostable DNA polymerases as an enhancing factor (Pfu Turbo DNA polymerase). By scavenging dUTP from the PCR mixture, the thermostable enzyme enables synthesis of long DNA fragments (>10kb) [10]. In this study, a gene encoding the dUTPase from Thermococcus pacificus was identified, cloned and sequenced. Comparing the sequence of the encoded Tpa dUTPase to other archaeal dUTPases indicated that the gene encodes a Tpa dUTPase. The recombinant Tpa dUTPase expressed in E. coli was purified and biochemically characterized. We also demonstrated that recombinant Tpa dUTPase can improve the efficiency of PCR amplification by preventing incorporation of dUTP generated from dCTP during PCR.
Materials and methods
Results and discussion
Acknowledgements This work was supported by the Marine and Extreme Genome Research Center Program of the Ministry of Land, Transportation and Maritime Affairs, Republic of Korea.