Characterization and PCR Application of Family B DNA Polymerases from .

DNA polymerases from the hyperthermophilic Archaea have attracted considerable attention as PCR enzymes due to their high thermal stability and proofreading 3' → 5' exonuclease activity. This study is the first to report data concerning the purification and biochemical characteristics of the Tst DNA polymerase from . Both the wild type Tst(wt) DNA polymerase and its chimeric form containing the P36H substitution-which reduces the enzyme's affinity for the U-containing template and dUTP-and the DNA-binding domain Sso7d from were obtained and analyzed. It was shown that Tst(wt) could effectively amplify up to 6-kb DNA fragments, whereas TstP36H-Sso7d could amplify DNA fragments up to 15 kb. It was found that TstP36H-Sso7d has superior PCR efficiency compared to the commonly used DNA polymerase PfuV93Q-Sso7d. For the amplification of a 2-kb DNA fragment, TstP36H-Sso7d required less than 10 s of extension time, whereas for PfuV93Q-Sso7d, the extension time was no less than 30 s. Steady-state kinetic assays revealed that the dNTP-binding affinity was the same for TstP36H-Sso7d and PfuV93Q-Sso7d, whereas the maximum rate of dNTP incorporation, , was two orders of magnitude higher for TstP36H-Sso7d. Moreover, the incorporation of incorrect dNTP was not observed for TstP36H-Sso7d up to 56 °C, whereas for PfuV93Q-Sso7d, the extension of primer with incorrect dNTP was observed at 37 °C, supporting higher fidelity of TstP36H-Sso7d. The obtained data suggest that TstP36H-Sso7d may be a good candidate for high-fidelity DNA amplification.