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Improved PCR performance using mutant Tpa-S DNA polymerases from the hyperthermophilic archaeon Thermococcus pacificus

Paper ID Volume ID Publish Year Pages File Format Full-Text
23307 43431 2013 8 PDF Available
Title
Improved PCR performance using mutant Tpa-S DNA polymerases from the hyperthermophilic archaeon Thermococcus pacificus
Abstract

We previously reported that Tpa-S DNA polymerase (constructed via fusion of the Sso7d DNA binding protein to the C-terminus of Thermococcus pacificus (Tpa) DNA polymerase) is more efficient in long and rapid PCR than wild-type Tpa, Taq, or Pfu DNA polymerases. However, Tpa-S DNA polymerase had a low yield of PCR products compared with commercialized Taq or Pfu DNA polymerases. To improve the yield of PCR products, mutant Tpa-S DNA polymerases were created via site-directed mutagenesis. In this study, we have targeted the N213 residue in the Exo II motif and the K501 residue in the Pol III motif. The mutant Tpa-S DNA polymerases showed enhanced PCR yields compared to that of the Tpa-S DNA polymerase. Specifically, the double mutant Tpa-S N213D/K501R DNA polymerase had an approximately three-fold increase in the yield of 8–10 kb PCR products over that of the Tpa-S DNA polymerase, and catalyzed amplification of a 12 kb PCR product using a lambda template with an extension time of 30 s. Even though the mutation is in the Exo II motif, the error rate of the double mutant Tpa-S N213D/K501R (2.79 × 10−5) was nearly the same as that seen in the Pfu DNA polymerase (2.70 × 10−5).

► Thermococcus pacificus-S N213D/K501R (Tpa-S N213D/K501R) gene contained open reading frame that encoded 840 amino acid residues. ► The molecular mass of Tpa-S N213D/K501R DNA polymerase was about 97 kDa. ► Tpa-S N213D/K501R DNA polymerase could amplify 12 kb target template at 30 s extension time per cycle. ► Each 213D and 501R point mutation had positive effect on the processivity of DNA polymerase. 501R point mutation had also upward fidelity tendency. ► Because Tpa-S N213D/K501R DNA polymerase had 3′ → 5′ exonuclease activity, it had practically same fidelity with Pfu DNA polymerase.

Keywords
Thermococcus pacificus-S (Tpa-S); Tpa-S DNA polymerase; Tpa-S N213D/K501R DNA polymerase; Polymerase chain reaction (PCR); PCR amplification; Fidelity
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Improved PCR performance using mutant Tpa-S DNA polymerases from the hyperthermophilic archaeon Thermococcus pacificus
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 164, Issue 2, March 2013, Pages 363–370
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