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Biotechnology Letters

, Volume 33, Issue 2, pp 339–346 | Cite as

Cloning, expression, and PCR application of DNA polymerase from the hyperthermophilic archaeon, Thermococcus celer

  • Kee Pum Kim
  • Heejin Bae
  • In Hye Kim
  • Suk-Tae Kwon
Original Research Paper

Abstract

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.

Keywords

DNA polymerase PCR improvement Thermococcus celer 

Notes

Acknowledgment

This work was supported by the Marine and Extreme Genome Research Center Program of the Ministry of Land, Transportation and Maritime Affairs, Republic of Korea.

References

  1. Barnes WM (1994) PCR amplification of up to 35-kb DNA with high fidelity and high yield from λ bacteriophage templates. Proc Natl Acad Sci USA 91:2216–2220CrossRefPubMedGoogle Scholar
  2. Braithwaite DK, Ito J (1993) Compilation, alignment, and phylogenetic relationships of DNA polymerases. Nucleic Acids Res 21:787–802CrossRefPubMedGoogle Scholar
  3. Cariello NF, Swenberg JA, Skopek TR (1991) Fidelity of Thermococcus litoralis DNA polymerase (Vent) in PCR determined by denaturing gradient gel electrophoresis. Nucleic Acids Res 19:4193–4198CrossRefPubMedGoogle Scholar
  4. Chien A, Edgar DB, Trela JM (1976) Deoxyribonucleic acid polymerase from the extreme thermophile Thermus aquaticus. J Bacteriol 127:1550–1557PubMedGoogle Scholar
  5. Choi JJ, Nam KH, Min B, Kim SJ, Söll D, Kwon ST (2006) Protein trans-splicing and characterization of a split family B-type DNA polymerase from the hyperthermophilic archaeal parasite Nanoarchaeum equitans. J Mol Biol 356:1093–1106CrossRefPubMedGoogle Scholar
  6. Choi JJ, Song JG, Nam KH, Lee JI, Bae H, Kim GA, Sun Y, Kwon ST (2008) Unique substrate spectrum and PCR application of Nanoarchaeum equitans family B DNA polymerase. Appl Environ Microbiol 74:6563–6569CrossRefPubMedGoogle Scholar
  7. Cline J, Braman JC, Hogrefe HH (1996) PCR fidelity of Pfu DNA polymerase and other thermostable DNA polymerases. Nucleic Acids Res 24:3546–3551CrossRefPubMedGoogle Scholar
  8. Kim YJ, Lee HS, Bae SS, Jeon JH, Lim JK, Cho Y, Nam KH, Kang SG, Kim SJ, Kwon ST (2007) Cloning, purification, and characterization of a new DNA polymerase from a hyperthermophilic archaeon, Thermococcus sp. NA1. J Microbiol Biotechnol 17:1090–1097PubMedGoogle Scholar
  9. Lehman IR, Bessman MJ, Simms ES, Kornberg A (1958) Enzymatic synthesis of deoxyribonucleic acid. I. Preparation of substrates and partial purification of an enzyme from Escherichia coli. J Biol Chem 233:163–170PubMedGoogle Scholar
  10. Lundberg KS, Shoemaker DD, Adams MW, Short JM, Sorge JA, Mathur EJ (1991) High-fidelity amplification using a thermostable DNA polymerase isolated from Pyrococcus furiosus. Gene 108:1–6CrossRefPubMedGoogle Scholar
  11. Mattila P, Korpela J, Tenkanen T, Pitkaenen K (1991) Fidelity of DNA synthesis by the Thermococcus litoralis DNA polymerase—an extremely heat stable enzyme with proofreading activity. Nucleic Acids Res 19:4967–4973CrossRefPubMedGoogle Scholar
  12. Nishioka M, Mizuguchi H, Fujiwara S, Komatsubara S, Kitabayashi M, Uemura H, Takagi M, Imanaka T (2001) Long and accurate PCR with a mixture of KOD DNA polymerase and its exonuclease deficient mutant enzyme. J Biotechnol 88:141–149CrossRefPubMedGoogle Scholar
  13. Ohmori H, Friedberg E, Fuchs R, Goodman M, Hanaoka F, Hinkle D, Kunkel T, Lawrence C, Livneh Z, Nohmi T, Prakash L, Prakash S, Todo T, Walker G, Wang Z, Woodgate R (2001) The Y-family of DNA polymerases. Mol Cell 8:7–8CrossRefPubMedGoogle Scholar
  14. Sanger F, Coulson AR, Hong GF, Hill DF, Petersen GB (1982) Nucleotide sequence of bacteriophage lambda DNA. J Mol Biol 162:729–773CrossRefPubMedGoogle Scholar
  15. Zillig W, Holz I, Janekovic D, Schaefer W, Reiter WD (1983) The archaebacterium Thermococcus celer represents a novel genus within the thermophilic branch of the archaebacteria. Syst Appl Microbiol 4:88–94Google Scholar

Copyright information

© Springer Science+Business Media B.V. 2010

Authors and Affiliations

  • Kee Pum Kim
    • 1
  • Heejin Bae
    • 1
  • In Hye Kim
    • 1
  • Suk-Tae Kwon
    • 1
  1. 1.Department of Genetic EngineeringSungkyunkwan UniversitySuwonRepublic of Korea

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