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 8-10kb PCR products over that of the Tpa-S DNA polymerase, and catalyzed amplification of a 12kb PCR product using a lambda template with an extension time of 30s. 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)).
authors: Department of Biotechnology and Bioengineering, Sungkyunkwan University, Jangan-gu, Suwon, Republic of Korea.
authors: Hyewoo
authors: H
authors: Ppyun
authors: Inhye
authors: I
authors: Kim
authors: Sung Suk
authors: SS
authors: Cho
authors: Kang Jin
authors: KJ
authors: Seo
authors: Keejung
authors: K
authors: Yoon
authors: Suk-Tae
authors: ST
authors: Kwon
mesh_terms: D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D015636:Magnesium Chloride / Q000737:chemistry; D009154:Mutation; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods*; D011189:Potassium Chloride / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019712:Thermococcus / Q000201:enzymology* / Q000235:genetics*
chemical_list: D011994:Recombinant Proteins; D015636:Magnesium Chloride; D011189:Potassium Chloride; D004247:DNA; D004259:DNA-Directed DNA Polymerase