Characterization of DNA polymerase from the hyperthermophilic archaeon Thermococcus marinus and its application to PCR.
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).
Department of Genetic Engineering, Sungkyunkwan University, Cheoncheon-dong, Jangan-gu, Suwon, Korea.
Heejin
H
Bae
Kee Pum
KP
Kim
Jong Il
JI
Lee
Jae-Geun
JG
Song
Eui-Joon
EJ
Kil
Joong Su
JS
Kim
Suk-Tae
ST
Kwon
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D017931:DNA Primers / Q000737:chemistry; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry*; D005092:Exonucleases / Q000378:metabolism; D020869:Gene Expression Profiling; D019766:Genes, Archaeal; D006863:Hydrogen-Ion Concentration; D008957:Models, Genetic; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010802:Phylogeny; D016133:Polymerase Chain Reaction / Q000295:instrumentation* / Q000379:methods*; D017422:Sequence Analysis, DNA; D019712:Thermococcus / Q000201:enzymology*
D017931:DNA Primers; D004259:DNA-Directed DNA Polymerase; D005092:Exonucleases