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Title: Processivity and thermostability of archaeal DNA polymerases : application in PCR
Author: Kinsman, Thomas Stephen
ISNI:       0000 0004 5356 7916
Awarding Body: University of Newcastle Upon Tyne
Current Institution: University of Newcastle upon Tyne
Date of Award: 2014
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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.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available