Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Cloning, Purification, and Characterization of a New DNA Polymerase from a Hyperthermophilic Archaeon, Thermococcus sp. NA1

  • Kim, Yun-Jae (Korea Ocean Research and Development Institute) ;
  • Lee, Hyun-Sook (Korea Ocean Research and Development Institute) ;
  • Bae, Seung-Seob (Korea Ocean Research and Development Institute) ;
  • Jeon, Jeong-Ho (Korea Ocean Research and Development Institute) ;
  • Lim, Jae-Kyu (Korea Ocean Research and Development Institute) ;
  • Cho, Yon-A (Korea Ocean Research and Development Institute) ;
  • Nam, Ki-Hoon (Department of Genetic Engineering, Sungkyunkwan University) ;
  • Kang, Sung-Gyun (Korea Ocean Research and Development Institute) ;
  • Kim, Sang-Jin (Korea Ocean Research and Development Institute) ;
  • Kwon, Suk-Tae (Department of Genetic Engineering, Sungkyunkwan University) ;
  • Lee, Jung-Hyun (Korea Ocean Research and Development Institute)
  • Published : 2007.07.31
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Abstract

Genomic analysis of Thermococcus sp. NA1 revealed the presence of a 3,927-base-pair (bp) family B-type DNA polymerase gene, TNA1_pol. TNA1_pol, without its intein, was overexpressed in Escherichia coli, purified using metal affinity chromatography, and characterized. TNA1_pol activity was optimal at pH 7.5 and $75^{\circ}C$. TNA1_pol was highly thermostable, with a half-life of 3.5h at $100^{\circ}C$ and 12.5h at $95^{\circ}C$. Polymerase chain reaction parameters of TNA1_pol such as error-rate, processivity, and extension rate were measured in comparison with rTaq, Pfu, and KOD DNA polymerases. TNA1_pol averaged one incorrect bp every 4.45 kilobases (kb), and had a processivity of 150 nucleotides (nt) and an extension rate of 60 bases/s. Thus, TNA1_pol has a much faster elongation rate than Pfu DNA polymerase with 7-fold higher fidelity than that of rTaq.

Keywords

References

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