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Enhanced PCR efficiency of high-fidelity DNA polymerase from Thermococcus waiotapuensis

Paper ID Volume ID Publish Year Pages File Format Full-Text
17166 42648 2014 7 PDF Available
Title
Enhanced PCR efficiency of high-fidelity DNA polymerase from Thermococcus waiotapuensis
Abstract

•We created mutant Twa H633R DNA polymerase using site-directed mutagenesis.•Twa H633R mutant showed an improved processivity compared to wild-type.•Twa H633R mutant showed an improved extension rate compared to wild-type.•Twa H633R mutant showed an improved PCR efficiency without the loss of fidelity.

Twa DNA polymerase from hyperthermophilic archaeon Thermococcus waiotapuensis has exceedingly high fidelity among family B DNA polymerases. However, Twa DNA polymerase has significant shortcomings in terms of a low extension rate and poor processivity. To resolve these weaknesses, we focused on two amino acid residues (N565 and H633) in the palm and thumb subdomains of the Twa DNA polymerase. These two residues were replaced by site-directed mutagenesis and the enzymatic properties of the mutants were analyzed. Here, Twa H633R DNA polymerase showed significantly improved polymerase function compared to wild-type Twa DNA polymerase in terms of processivity (2-fold), extension rate (1.5-fold) and PCR efficiency. Kinetic analysis using DNA as a template revealed that the kcat value of the Twa H633R mutant was similar to that of wild-type, but the Km of the Twa H633R mutant was about 1.6-fold lower than that of the wild-type. These results showed that the Arg residue substitution at H633 located in the thumb subdomain has a positive effect on processivity, extension rate and PCR efficiency, suggesting that the Twa H633R mutant allows a conformational change for easy access of the primer-template to the binding site of the polymerase domain.

Keywords
Thermococcus waiotapuensis (Twa); Twa DNA polymerase; Twa H633R DNA polymerase; PCR amplification
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Publisher
Database: Elsevier - ScienceDirect
Journal: Enzyme and Microbial Technology - Volume 63, September 2014, Pages 39–45
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us