Evolution of PCR Enzymes (towards a better PCR system based on a KOD DNA polymerase)

摘要

Taq DNA polymerase from Thermus aquaticus and Tth DNA polymerase from Thermus thermophilus are thermostable DNA polymerases conventionally used in PCR (polymerase chain reaction) and they are classified in pol I like bacterial DNA polymerase family (family A). However, recently archaeal DNA polymerases classified in a-like DNA polymerase family (family B DNA polymerase) from Pyrococcus furiosus, Pyrococcus GB-D, Thermococcus litoralis are often used in PCR because of their high fidelity in DNA synthesis based on 3 ' - 5 ' exonuclease activity for proofreading of misincorporated nucleotides. Indeed high fidelity is ideal for PCR but these family B polymerases often require longer reaction time (at least two minutes) because of their low elongation speed. We have recently reported a new thermostable family B polymerase, KOD DNA polymerase from hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 (previously Pyrococcus kodakaraensis KOD1), which is an efficient PCR enzyme with high fidelity and extension rate. In this article, development of PCR enzymes including (1) Characterization of a new PCR enzyme, KOD DNA polymerase, (2) Engineering of a new long and accurate (LA) PCR enzyme, and (3) Improvement of PCR by neutralizing monoclonal antibodies, will be reviewed. [References: 23]