호열성 dUTPase의 특성과 PCR 응용

Title
호열성 dUTPase의 특성과 PCR 응용
Alternative Title
Characterization of dUTPase from the Hyperthermophilic Archaeon Thermococcus sp. NA1 and its Application to PCR
Author(s)
조요나; 이현숙; 김윤재; 배승섭; 임재규; 강성균; 이정현
KIOST Author(s)
Lee, Hyun Sook(이현숙)Kang, Sung Gyun(강성균)Lee, Jung Hyun(이정현)
Publication Year
2006-05-03
Abstract
Genomic analysis of a hyperthermophilic archaeon Thermococcus sp. NA1(TNA1) revealed the presence of an open reading frame consisting of 468 bp similar to dUTPases from Pyrococcus woesei and P. furiosus (83% identity and 93% similarity). TNA1 dUTPase has five motifs conserved in all dUTPases from eukarya and prokarya origins. The dUTPase gene was cloned and expressed in Escherichia coli. The purified protein behaved as a dimer in gel filtration and was able to hydrolyse both dUTP and dCTP. The optimum activity toward dUTP occurred at 80°C and pH 8.0. Moreover, the enzyme activity was highly dependent on Mg2+ concentration with optimum activity at 1mM to 5mM which is usually used at PCR amplification. TNA1 dUTPase activity was a little affected by KCl and (NH4)2SO4, while Tritoxn X-100 slightly increased dUTPase activity. The enzyme displayed thermostability with half-life (t1/2) of 170 min at 95°C. TNA1 dUTPase enhanced the PCR in a broader range of target lengths in combination with TNA1 DNA polymerase. Moreover, TNA1 dUTPase increased the yield of PCR products as much as other archaeal DNA polymerases.
URI
https://sciwatch.kiost.ac.kr/handle/2020.kiost/31111
Bibliographic Citation
2006(봄)한국미생물학회, pp.197, 2006
Publisher
한국미생물학회
Type
Conference
Language
English
Publisher
한국미생물학회
Related Researcher
Research Interests

marine biotechnology,molecular microbiology,해양생명공학,분자미생물학

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