title: Enhancing the processivity of a family B-type DNA polymerase of Thermococcus onnurineus and application to long PCR.
abstract: Mechanisms that allow replicative DNA polymerases to attain high processivity are often specific to a given polymerase and cannot be generalised to others. Amplification efficiency is lower in family B-type DNA polymerases than in family A-type (Taq) polymerases because of their strong 3'-5' exonuclease-activity. Here, we have red the exonuclease domain of the Thermococcus onnurineus NA1 (TNA1) DNA polymerase, especially Asn210 to Asp215 residues in Exo II motif (NXXXFD), to improve the processivity. N213D mutant protein had higher processivity and extension rate than the wild-type TNA1 DNA polymerase, retaining a lower mutation frequency than recombinant Taq DNA polymerase. Consequently, the N213D mutant could amplify target DNA up to 13.5 kb in length from human genomic DNA and 16.2 kb in length from human mitochondrial DNA while wild-type TNA1 amplified target DNA of 2.7 kb in length from human genomic DNA.
authors.affiliation: Korea Institute of Ocean Science and Technology, Ansan, 426-744, Korea, bio1213@kiost.ac.
authors.forename: Yun Jae
authors.initials: YJ
authors.lastname: Kim
authors.forename: Hyun Sook
authors.initials: HS
authors.lastname: Lee
authors.forename: Suk-Tae
authors.initials: ST
authors.lastname: Kwon
authors.forename: Jung-Hyun
authors.initials: JH
authors.lastname: Lee
authors.forename: Sung Gyun
authors.initials: SG
authors.lastname: Kang
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*; D019712:Thermococcus / Q000201:enzymology / Q000235:genetics*
chemical_list: D001426:Bacterial Proteins; D004259:DNA-Directed DNA Polymerase