Characterization of a family B DNA polymerase from Thermococcus barophilus Ch5 and its application for long and accurate PCR SCIE SCOPUS

Cited 6 time in WEB OF SCIENCE Cited 6 time in Scopus
Title
Characterization of a family B DNA polymerase from Thermococcus barophilus Ch5 and its application for long and accurate PCR
Author(s)
Kwon, Kyung-Min; Kang, Sung Gyun; Sokolova, Tatyana G.; Cho, Sung Suk; Kim, Yun Jae; Kim, Cheorl-Ho; Kwon, Suk-Tae
KIOST Author(s)
Kang, Sung Gyun(강성균)
Publication Year
2016-05
Abstract
The family B DNA polymerase gene from the euryarchaeon Thermococcus barophilus Ch5 (Tba5) contains an open reading frame of 6198 base pairs that encodes 2065 amino acid residues. The gene is split by three inteins that must be spliced out to form the mature DNA polymerase. A Tba5 DNA polymerase gene without inteins (genetically intein-spliced) was expressed under the control of the pET-28b(+)T7lac promoter in E. coli Rosetta 2(DE3)pLysS cells. The molecular mass of the purified Tba5 DNA polymerase was about 90 kDa consistent with the 90,470 Da molecular mass calculated based on the 776 amino acid sequence. The optimal pH for Tba5 DNA polymerase activity was 7.5 and the optimal temperature was 70-75 degrees C. The enzyme possessed 3' -> 5' exonuclease activity and was activated by magnesium ions. PCR amplification using Tba5 DNA polymerase enables high-yield for 1- to 6-kb target DNA products, while 8- to 10-kb target DNA products were amplified at low or inefficient levels. To simultaneously improve product yield and amplification fidelity, Tba5 plus DNA polymerase mixtures were constituted with various amounts of Tba5 DNA polymerase mixed with Taq DNA polymerase. The Tba5 plus DNA polymerase mixtures robustly amplified up to 25-kb lambda DNA fragments. In addition, the PCR error rate of Tba5 plus3 and Tba5 plus4 mixtures were much lower than those of wild-type Tba5 DNA polymerase, Pfu DNA polymerase, Taq DNA polymerase, and Pfu plus DNA polymerase. (C) 2016 Elsevier Inc. All rights reserved.
ISSN
0141-0229
URI
https://sciwatch.kiost.ac.kr/handle/2020.kiost/2197
DOI
10.1016/j.enzmictec.2016.02.004
Bibliographic Citation
ENZYME AND MICROBIAL TECHNOLOGY, v.86, pp.117 - 126, 2016
Publisher
ELSEVIER SCIENCE INC
Subject
HIGH-FIDELITY; NANOARCHAEUM-EQUITANS; AMPLIFICATION; PURIFICATION; PROCESSIVITY; REPLICATION; EXPRESSION; EFFICIENCY; CLONING
Keywords
Thermococcus barophilus Ch5 DNA polymerase (Tba5); PCR amplification; Fidelity; Long and accurate PCR (LA PCR)
Type
Article
Language
English
Document Type
Article
Publisher
ELSEVIER SCIENCE INC
Related Researcher
Research Interests

marine biotechnology,microbiology,해양생명공학,미생물학

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