Characterization of a family B DNA polymerase from Thermococcus eurythermalis A501 and its application in PCR / 生物工程学报
Chinese Journal of Biotechnology
;
(12): 807-819, 2022.
Article
in Chinese
| WPRIM
| ID: wpr-927746
ABSTRACT
DNA polymerases are widely used in PCR and play important roles in life science research and related fields. Development of high-performance DNA polymerases is of great commercial interest as the current commercial DNA polymerases could not fully satisfy the requirements of scientific research. In this study, we cloned and expressed a family B DNA polymerase (NCBI accession number TEU_RS04875) from Thermococcus eurythermalis A501, characterized its enzymatic property and evaluated its application in PCR. The recombinant Teu-PolB was expressed in E. coli and purified with affinity chromatography and ion-exchange chromatography. The enzymatic properties of Teu-PolB were characterized using fluorescence-labeled oligonucleotides as substrates. The application potential of Teu-PolB in PCR was evaluated using the phage λ genomic DNA as a template. Teu-PolB has DNA polymerase and 3'→5' exonuclease activities, and is highly thermostable with a half-life of 2 h at 98 ℃. The most suitable PCR buffer is consisted of 50 mmol/L Tris-HCl pH 8.0, 2.5 mmol/L MgCl2, 60 mmol/L KCl, 10 mmol/L (NH4)2SO4, 0.015% Triton X-100 and 0.01% BSA, and the optimal extension temperature is 68 ℃. Under the optimized conditions, a 4 kb target fragment was successfully amplified with an extension rate of 2 kb/min. The yield of the Teu-PolB amplified-DNA was lower than that of Taq DNA polymerase, but its extension rate and fidelity was higher than that of Taq and Pfu DNA polymerases. The biochemical properties of Teu-PolB demonstrate that this enzyme can be used in PCR amplification with high thermostability, good salt tolerance, high extension rate and high fidelity.
Full text:
Available
Index:
WPRIM (Western Pacific)
Main subject:
Temperature
/
Polymerase Chain Reaction
/
Thermococcus
/
DNA-Directed DNA Polymerase
/
Escherichia coli
Language:
Chinese
Journal:
Chinese Journal of Biotechnology
Year:
2022
Type:
Article
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