Improved PCR performance using mutant Tpa-S DNA polymerases from the hyperthermophilic archaeon Thermococcus pacificus

► Thermococcus pacificus-S N213D/K501R (Tpa-S N213D/K501R) gene contained open reading frame that encoded 840 amino acid residues. ► The molecular mass of Tpa-S N213D/K501R DNA polymerase was about 97kDa. ► Tpa-S N213D/K501R DNA polymerase could amplify 12kb target template at 30s extension time per cycle. ► Each 213D and 501R point mutation had positive effect on the processivity of DNA polymerase. 501R point mutation had also upward fidelity tendency. ► Because Tpa-S N213D/K501R DNA polymerase had 3′→5′ exonuclease activity, it had practically same fidelity with Pfu DNA polymerase. We previously reported that Tpa-S DNA polymerase (constructed via fusion of the Sso7d DNA binding protein to the C-terminus of Thermococcus pacificus (Tpa) DNA polymerase) is more efficient in long and rapid PCR than wild-type Tpa, Taq, or Pfu DNA polymerases. However, Tpa-S DNA polymerase had a low yield of PCR products compared with commercialized Taq or Pfu DNA polymerases. To improve the yield of PCR products, mutant Tpa-S DNA polymerases were created via site-directed mutagenesis. In this study, we have targeted the N213 residue in the Exo II motif and the K501 residue in the Pol III motif. The mutant Tpa-S DNA polymerases showed enhanced PCR yields compared to that of the Tpa-S DNA polymerase. Specifically, the double mutant Tpa-S N213D/K501R DNA polymerase had an approximately three-fold increase in the yield of 8–10kb PCR products over that of the Tpa-S DNA polymerase, and catalyzed amplification of a 12kb PCR product using a lambda template with an extension time of 30s. Even though the mutation is in the Exo II motif, the error rate of the double mutant Tpa-S N213D/K501R (2.79×10−5) was nearly the same as that seen in the Pfu DNA polymerase (2.70×10−5).