Biotechnology Letters

, Volume 36, Issue 5, pp 985–992 | Cite as

Enhancing the processivity of a family B-type DNA polymerase of Thermococcus onnurineus and application to long PCR

  • Yun Jae Kim
  • Hyun Sook Lee
  • Suk-Tae Kwon
  • Jung-Hyun Lee
  • Sung Gyun KangEmail author
Original Research Paper


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.


DNA polymerase Error rate 3′–5′ Exonuclease domain Processivity Thermococcus DNA polymerase TNA1 DNA polymerase 



This work was supported by the KIOST in-house Program (PE98983, PE98993), the Marine and Extreme Genome Research Center Program and the Development of Biohydrogen Production Technology using Hyperthermophilic Archaea Program of the Ministry of Ocean and Fisheries, Republic of Korea.

Supplementary material

10529_2013_1441_MOESM1_ESM.docx (22 kb)
Supplementary material 1 (DOCX 21 kb)


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Copyright information

© Springer Science+Business Media Dordrecht 2013

Authors and Affiliations

  • Yun Jae Kim
    • 1
    • 2
  • Hyun Sook Lee
    • 1
    • 2
  • Suk-Tae Kwon
    • 3
  • Jung-Hyun Lee
    • 1
    • 2
  • Sung Gyun Kang
    • 1
    • 2
    Email author
  1. 1.Korea Institute of Ocean Science and TechnologyAnsanKorea
  2. 2.Department of Marine BiotechnologyUniversity of Science and TechnologyTaejonKorea
  3. 3.Department of Genetic EngineeringSungkyunkwan UniversitySuwonKorea

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