title: Characterization of DNA polymerase from Pyrococcus sp. strain KOD1 and its application to PCR.
abstract: The DNA polymerase gene from the archaeon Pyrococcus sp. strain KOD1 (KOD DNA polymerase) contains a long open reading frame of 5,013 bases that encodes 1,671 amino acid residues (GenBank accession no. D29671). Similarity analysis revealed that the DNA polymerase contained a putative 3'-5' exonuclease activity and two in-frame intervening sequences of 1,080 bp (360 amino acids; KOD pol intein-1) and 1,611 bp (537 amino acids; KOD pol intein-2), which are located in the middle of regions conserved among eukaryotic and archaeal alpha-like DNA polymerases. The mature form of the DNA polymerase gene was expressed in Escherichia coli, and the recombinant enzyme was purified and characterized. 3'-5' exonuclease activity was confirmed, and although KOD DNA polymerase's optimum temperature (75 degrees C) and mutation frequency (3.5 x 10(-3)) were similar to those of a DNA polymerase from Pyrococcus furiosus (Pfu DNA polymerase), the KOD DNA polymerase exhibited an extension rate (100 to 130 nucleotides/s) 5 times higher and a processivity (persistence of sequential nucleotide polymerization) 10 to 15 times higher than those of Pfu DNA polymerase. These characteristics enabled the KOD DNA polymerase to perform a more accurate PCR in a shorter reaction time.
authors.affiliation: Department of Biotechnology, Graduate School of Engineering, Osaka University, Japan.
authors.forename: M
authors.initials: M
authors.lastname: Takagi
authors.forename: M
authors.initials: M
authors.lastname: Nishioka
authors.forename: H
authors.initials: H
authors.lastname: Kakihara
authors.forename: M
authors.initials: M
authors.lastname: Kitabayashi
authors.forename: H
authors.initials: H
authors.lastname: Inoue
authors.forename: B
authors.initials: B
authors.lastname: Kawakami
authors.forename: M
authors.initials: M
authors.lastname: Oka
authors.forename: T
authors.initials: T
authors.lastname: Imanaka
mesh_terms: D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000235:genetics; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D019711:Pyrococcus / Q000201:enzymology*
chemical_list: D004259:DNA-Directed DNA Polymerase