DNA polymerase hybrids derived from the family-B enzymes of Pyrococcus furiosus and Thermococcus kodakarensis: improving performance in the polymerase chain reaction
Ashraf M. Elshawadfy, Brian J. Keith, H'Ng Ee Ooi, Thomas Kinsman, Pauline Heslop, Bernard A. Connolly
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.
Pharmacology, Toxicology and Pharmaceutical Science
2
2%
Immunology and Microbiology
2
2%
Other
3
3%
Unknown
23
27%
Attention Score in Context
Attention Score in Context
This research output has an Altmetric Attention Score of 12. This is our high-level measure of the quality and quantity of online attention that it has received. This Attention Score, as well as the ranking and number of research outputs shown below, was calculated when the research output was last mentioned on 19 October 2022.
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#2,767,472
of 23,555,482 outputs
Outputs from Frontiers in Microbiology
#2,368
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Outputs of similar age
#28,139
of 228,293 outputs
Outputs of similar age from Frontiers in Microbiology
#20
of 165 outputs
Altmetric has tracked 23,555,482 research outputs across all sources so far. Compared to these this one has done well and is in the 88th percentile: it's in the top 25% of all research outputs ever tracked by Altmetric.
So far Altmetric has tracked 26,044 research outputs from this source. They typically receive a little more attention than average, with a mean Attention Score of 6.4. This one has done particularly well, scoring higher than 90% of its peers.
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We're also able to compare this research output to 165 others from the same source and published within six weeks on either side of this one. This one has done well, scoring higher than 87% of its contemporaries.
