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Characterization and PCR application of a new high-fidelity DNA polymerase from Thermococcus waiotapuensis

Paper ID Volume ID Publish Year Pages File Format Full-Text
17186 42650 2012 8 PDF Available
Title
Characterization and PCR application of a new high-fidelity DNA polymerase from Thermococcus waiotapuensis
Abstract

The family B DNA polymerase gene from the euryarchaeon Thermococcus waiotapuensis (Twa) contains an open reading frame of 4404 bases that encodes 1467 amino acid residues. The gene is split by two intein-coding sequences that forms a continuous open reading frame with the three polymerase exteins. Twa DNA polymerase genes with (whole gene) and without (genetically intein-spliced) inteins were expressed in Escherichia coli Rosetta(DE3)pLysS. The inteins of the expressed whole gene were easily spliced during purification. The molecular mass of the purified Twa DNA polymerase was about 90 kDa, as estimated by SDS-PAGE. The optimal pH for Twa DNA polymerase activity was 6.0 and the optimal temperature was 75 °C. The enzyme was activated by magnesium ions. The half-life of the enzyme at 99 °C was about 4 h. The optimal buffer for PCR with Twa DNA polymerase was 50 mM Tris–HCl (pH 8.2), 2.0 mM MgCl2, 30 mM KCl, 2.0 mM (NH4)2SO4, 0.01% Triton X-100, and 0.005% BSA. The PCR fidelity of Twa DNA polymerase was higher than Pfu, KOD and Vent DNA polymerases. A ratio of 15:1 Taq:Twa DNA polymerase efficiently facilitated long-range PCR.

► Thermococcus waiotapuensis (Twa) DNA polymerase gene contains an open reading frame of 4404 bases that encodes 1467 amino acid residues. ► The molecular mass of the purified Twa DNA polymerase was about 90 kDa. ► The PCR fidelity of Twa DNA polymerase was higher than Pfu, KOD and Vent DNA polymerases. ► A ratio of 15:1 Taq:Twa DNA polymerase efficiently facilitated long-range PCR.

Keywords
Thermococcus waiotapuensis (Twa); Twa DNA polymerase; Polymerase chain reaction; Fidelity
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Characterization and PCR application of a new high-fidelity DNA polymerase from Thermococcus waiotapuensis
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Publisher
Database: Elsevier - ScienceDirect
Journal: Enzyme and Microbial Technology - Volume 51, Issues 6–7, 10 December 2012, Pages 334–341
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