Characterization of a dUTPase from the hyperthermophilic archaeon Thermococcus onnurineus NA1 and its application in polymerase chain reaction amplification SCIE SCOPUS

Cited 9 time in WEB OF SCIENCE Cited 10 time in Scopus
Title
Characterization of a dUTPase from the hyperthermophilic archaeon Thermococcus onnurineus NA1 and its application in polymerase chain reaction amplification
Author(s)
Cho, Y.; Lee, H. S.; Kim, Y. J.; Kang, S. G.; Kim, S.-J.; Lee, J.-H.
KIOST Author(s)
Lee, Hyun Sook(이현숙)Kang, Sung Gyun(강성균)Lee, Jung Hyun(이정현)
Publication Year
2007-08
Abstract
Genomic analysis of the hyperthermophilic archaeon Thermococcus onnurineus NA1 (TNA1) revealed the presence of a 471-bp open reading frame with 93% similarity to the dUTPase from Pyrococcus furiosus. The dUTPase-encoding gene was cloned and expressed in Escherichia coli. The purified protein hydrolyzed dUTP at about a 10-fold higher rate than dCTP. The protein behaved as a dimer in gel filtration chromatography, even though it contains five motifs that are conserved in all homotrimeric dUTPases. The dUTPase showed optimum activity at 80 degrees C and pH 8.0, and it was highly thermostable with a half-life (t (1/2)) of 170 min at 95 degrees C. The enzymatic activity of the dUTPase was largely unaffected by variations in MgCl2, KCl, (NH4)(2)SO4, and Triton X-100 concentrations, although it was reduced by bovine serum albumin. Addition of the dUTPase to polymerase chain reactions (PCRs) run with TNA1 DNA polymerase significantly increased product yield, overcoming the inhibitory effect of dUTP. Further, addition of the dUTPase allowed PCR amplification of targets up to 15 kb in length using TNA1 DNA polymerase. This enzyme also improved the PCR efficiency of other archaeal family B type DNA polymerases, including Pfu and KOD.
ISSN
1436-2228
URI
https://sciwatch.kiost.ac.kr/handle/2020.kiost/4668
DOI
10.1007/s10126-007-9002-8
Bibliographic Citation
MARINE BIOTECHNOLOGY, v.9, no.4, pp.450 - 458, 2007
Publisher
SPRINGER
Subject
THERMOSTABLE DNA-POLYMERASES; INFECTIOUS-ANEMIA VIRUS; ESCHERICHIA-COLI; CRYSTAL-STRUCTURE; LEISHMANIA-MAJOR; METAL-BINDING; HIGH-FIDELITY; ENZYME; PYROPHOSPHATASE; PURIFICATION
Keywords
DNA polymerase; dUTPase; PCR amplification; Thermococcus onnurineus NA1
Type
Article
Language
English
Document Type
Article
Publisher
SPRINGER
Related Researcher
Research Interests

marine biotechnology,molecular microbiology,해양생명공학,분자미생물학

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