Journal
BIOTECHNOLOGY LETTERS
Volume 36, Issue 5, Pages 985-992Publisher
SPRINGER
DOI: 10.1007/s10529-013-1441-x
Keywords
DNA polymerase; Error rate; 3 '-5 ' Exonuclease domain; Processivity; Thermococcus DNA polymerase; TNA1 DNA polymerase
Categories
Funding
- KIOST [PE98983, PE98993]
- Marine and Extreme Genome Research Center Program
- Ministry of Ocean and Fisheries, Republic of Korea
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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'aEuro5' 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.
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