Cloning, expression, and PCR application of DNA polymerase from the hyperthermophilic archaeon, Thermococcus celer.
The family B DNA polymerase gene was amplified from Thermococcus celer genomic DNA by using the degenerate primers and DNA walking PCR. The Tce DNA polymerase gene was cloned and sequenced. The gene contains an ORF of 2,325 bp encoding 774 amino acid residues with a calculated molecular weight of 89,788.9 kDa. The Tce DNA polymerase was purified by heat treatment and heparin column chromatography. The optimal conditions for PCR were determined. Long-range PCR and time-saving PCR were performed using various specific ratios of Taq and Tce DNA polymerases (Tce plus DNA polymerase). Tce plus DNA polymerase surpassed the PCR performance of Tce, Taq and Pfu DNA polymerases in terms of yield and efficiency.
Department of Genetic Engineering, Sungkyunkwan University, 300 Chunchun-dong, Jangan-gu, Suwon 440-746, Republic of Korea.
Kee Pum
KP
Kim
Heejin
H
Bae
In Hye
IH
Kim
Suk-Tae
ST
Kwon
D019843:Archaeal Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002846:Chromatography, Affinity / Q000379:methods; D003001:Cloning, Molecular; D019641:DNA, Archaeal / Q000737:chemistry / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006358:Hot Temperature; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016366:Open Reading Frames; D016133:Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA; D019712:Thermococcus / Q000201:enzymology* / Q000235:genetics
D019843:Archaeal Proteins; D019641:DNA, Archaeal; D004259:DNA-Directed DNA Polymerase