Characterization of DNA polymerase from the hyperthermophilic archaeon Thermococcus marinus and its application to PCR = 호열균인 Thermococcus marinus로 부터 DNA polymerase의 특성연구 및 PCR에 응용

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Title
Characterization of DNA polymerase from the hyperthermophilic archaeon Thermococcus marinus and its application to PCR = 호열균인 Thermococcus marinus로 부터 DNA polymerase의 특성연구 및 PCR에 응용
Author(s)
H Bae; K P Kim; J I Lee; J G Song; E J Kil; Joong Su Kim; S T Kwon
Bibliographic Citation
Extremophiles, vol. 13, no. 4, pp. 657-667
Publication Year
2009
Abstract
The family B DNA polymerase gene from the archaeon Thermococcus marinus (Tma) contains a long open reading frame of 3,939 bp that encodes 1,312 amino acid residues. The gene is split by one intervening sequence that forms a continuous open reading frame with the two polymerase exteins. In this study, the Tma DNA polymerase gene both with (precursor form) and without (mature form) its intein was expressed in Escherichia coli, purified by heat treatment and HiTrap Heparin HP column chromatography and characterized. Primary sequence analysis of the mature Tma polymerase showed high sequence identity with DNA polymerases in the genus Thermococcus. The expressed precursor form was easily spliced during purification steps. The molecular mass of the purified Tma DNA polymerases is about 90 kDa, as estimated by SDS-PAGE. Both Tma DNA polymerases showed the same properties. PCR performed with this enzyme was found to be optimal in the presence of 50 mM Tris-HCl (pH 8.4), 40 mM KCl, 12.5 mM (NH(4))(2)SO(4,) 2 mM MgCl(2,) 0.05% Triton X-100 and 0.0075% BSA. Furthermore, long-range PCR and time-saving PCR were performed using various specific ratios of Taq and Tma DNA polymerases (Tma plus DNA polymerase).
Keyword
DNA polymeraseThermococcus marinusPolymerase chain reactionPCR amplification rateFidelityArchaea
ISSN
1431-0651
Publisher
Springer
DOI
http://dx.doi.org/10.1007/s00792-009-0248-0
Type
Article
Appears in Collections:
Division of Bio Technology Innovation > SME Support Center > 1. Journal Articles
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