Characterization and PCR application of a thermostable DNA polymerase from Thermococcus pacificus

Abstract

The biochemical properties of the Thermococcus pacificus ( Tpa) DNA polymerase were determined to evaluate its feasibility for use in polymerase chain reaction (PCR) application. The Tpa DNA polymerase gene was expressed under the control of the T7 lac promoter in the pET-22b(+) plasmid in Escherichia coli BL21-CodonPlus(DE3)-RIL. The enzyme was then purified by heat treatment followed by two steps of column chromatography after which the optimum pH and temperature of the enzyme were determined to be pH 7.5 and 75 °C. The optimal PCR buffer for Tpa DNA polymerase consisted of 50 mM Tris–HCl (pH 8.4), 4 mM MgCl 2, and 10 mM KCl. Tpa DNA polymerase performed significantly more efficiently in PCR amplification than Taq or Pfu DNA polymerase. By fusing the Sulfolobus solfataricus DNA binding protein Sso7d to Tpa DNA polymerase, we obtained a fusion polymerase which exhibits profound advantages over unmodified Tpa DNA polymerase in PCR applications. Tpa DNA polymerase (2.04 × 10 −6) and Tpa-S DNA polymerase (2.20 × 10 −6) revealed a 5-fold higher fidelity than Taq DNA polymerase (12.13 × 10 −6).