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DNA polymerase hybrids derived from the family-B enzymes of Pyrococcus furiosus and Thermococcus kodakarensis: improving performance in the polymerase chain reaction

Lookup NU author(s): Brian Keith, Thomas Kinsman, Pauline Heslop, Professor Bernard Connolly

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This work is licensed under a Creative Commons Attribution 4.0 International License (CC BY 4.0).


Abstract

The polymerase chain reaction (PCR) is widely applied across the biosciences, with archaeal Family-B DNA polymerases being preferred, due to their high thermostability and fidelity. The enzyme from Pyrococcus furiosus (Pfu-Pol) is more frequently used than the similar protein from Thermococcus kodakarensis (Tkod-Pol), despite the latter having better PCR performance. Here the two polymerases have been comprehensively compared, confirming that Tkod-Pol: (1) extends primer-templates more rapidly; (2) has higher processivity; (3) demonstrates superior performance in normal and real time PCR. However, Tkod-Pol is less thermostable than Pfu-Pol and both enzymes have equal fidelities. To understand the favorable properties of Tkod-Pol, hybrid proteins have been prepared. Single, double and triple mutations were used to site arginines, present at the "forked-point" (the junction of the exonuclease and polymerase channels) of Tkod-Pol, at the corresponding locations in Pfu-Pol, slightly improving PCR performance. The Pfu-Pol thumb domain, responsible for double-stranded DNA binding, has been entirely replaced with that from Tkod-Pol, again giving better PCR properties. Combining the "forked-point" and thumb swap mutations resulted in a marked increase in PCR capability, maintenance of high fidelity and retention of the superior thermostability associated with Pfu-Pol. However, even the arginine/thumb swap mutant falls short of Tkod-Pol in PCR, suggesting further improvement within the Pfu-Pol framework is attainable. The significance of this work is the observation that improvements in PCR performance are easily attainable by blending elements from closely related archaeal polymerases, an approach that may, in future, be extended by using more polymerases from these organisms.


Publication metadata

Author(s): Elshawadfy AM, Keith BJ, Ooi HE, Kinsman T, Heslop P, Connolly BA

Publication type: Article

Publication status: Published

Journal: Frontiers in Microbiology

Year: 2014

Volume: 5

Print publication date: 27/05/2014

Online publication date: 27/05/2014

Acceptance date: 28/04/2014

Date deposited: 07/07/2015

ISSN (print): 1664-302X

ISSN (electronic): 2235-2988

Publisher: Frontiers Research Foundation

URL: http://dx.doi.org/10.3389/fmicb.2014.00224

DOI: 10.3389/fmicb.2014.00224


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