Processivity and thermostability of archaeal DNA polymerases :application in PCR

Abstract

The polymerase chain reaction (PCR) is one of the most widely used techniques in the biosciences, and has found extensive use in a variety of processes including gene cloning and mutagenesis. The PCR requires the use of a thermostable DNA polymerase that is able to tolerate the multiple heat/cool steps that occur during each cycle of the reaction. Archaeal family B DNA polymerases have found extensive use in this process, as in addition to their high thermostability they also contain a 3’-5’ exonuclease or proofreading activity, which increases the fidelity of replication. A polymerase that exhibits high processivity, defined as the number of nucleotides added per association with the DNA, is also desirable from a commercial perspective as it will reduce the amount of time taken to replicate any given amplicon. In this thesis, the processivity of a variety of commercially available archaeal Pol B enzymes is determined, which reveals significant differences in the processivity of polymerases closely related in sequence. The PCR performance of Pfu-Pol and Tkod-Pol, representing poorly and highly processive enzymes respectively is investigated, which reveals that Tkod-Pol is less efficient at replicating long amplicons (> 1000 bp) than Pfu-Pol, attributed to the increased thermostability of the latter. Based on this observation, an attempt is made to enhance the processivity of Pfu-Pol to improve the PCR performance of this enzyme.