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.
authors.affiliation: Department of Biotechnology and Bioengineering, Sungkyunkwan University, 300 Cheoncheon-dong, Jangan-gu, Suwon 440-746, Republic of Korea.
authors.forename: Sung Suk
authors.initials: SS
authors.lastname: Cho
authors.forename: Kee Pum
authors.initials: KP
authors.lastname: Kim
authors.forename: Kang Keun
authors.initials: KK
authors.lastname: Lee
authors.forename: Man-Hui
authors.initials: MH
authors.lastname: Youn
authors.forename: Suk-Tae
authors.initials: ST
authors.lastname: Kwon
mesh_terms: D019843:Archaeal Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D001483:Base Sequence; D001709:Biotechnology; D003001:Cloning, Molecular; D019641:DNA, Archaeal / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004795:Enzyme Stability; D019766:Genes, Archaeal; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D010802:Phylogeny; D016133:Polymerase Chain Reaction / Q000379:methods*; D017386:Sequence Homology, Amino Acid; D013696:Temperature; D019712:Thermococcus / Q000201:enzymology* / Q000235:genetics*
chemical_list: D019843:Archaeal Proteins; D019641:DNA, Archaeal; D004259:DNA-Directed DNA Polymerase