Characterization of a dITPase from the hyperthermophilic archaeon Thermococcus onnurineus NA1 and its application in PCR amplification SCIE SCOPUS

Cited 7 time in WEB OF SCIENCE Cited 7 time in Scopus
Title
Characterization of a dITPase from the hyperthermophilic archaeon Thermococcus onnurineus NA1 and its application in PCR amplification
Author(s)
Kim, Yun-Jae; Ryu, Yong-Gu; Lee, Hyun Sook; Cho, Yona; Kwon, Suk-Tae; Lee, Jung-Hyun; Kang, Sung Gyun
KIOST Author(s)
Lee, Hyun Sook(이현숙)Lee, Jung Hyun(이정현)Kang, Sung Gyun(강성균)
Publication Year
2008-06
Abstract
In this study, we found that deoxyinosine triphosphate (dITP) could inhibit polymerase chain reaction (PCR) amplification of various family B-type DNA polymerases, and 0.93% dITP was spontaneously generated from deoxyadenosine triphosphate during PCR amplification. Thus, it was hypothesized that the generated dITP might have negative effect on PCR amplification of family B-type DNA polymerases. To overcome the inhibitory effect of dITP during PCR amplification, a dITP pyrophosphatase (dITPase) from Thermococcus onnurineus NA1 was applied to PCR amplification. Genomic analysis of the hyperthermophilic archaeon T. onnurineus NA1 revealed the presence of a 555-bp open reading frame with 48% similarity to HAM1-like dITPase from Methanocaldococcus jannaschii DSM2661 (NP_247195). The dITPase-encoding gene was cloned and expressed in Escherichia coli. The purified protein hydrolyzed dITP, not deoxyuridine triphosphate. Addition of the purified protein to PCR reactions using DNA polymerases from T. onnurineus NA1 and Pyrococcus furiosus significantly increased product yield, overcoming the inhibitory effect of dITP. This study shows the first representation that removing dITP using a dITPase enhances the PCR amplification yield of family B-type DNA polymerase.
ISSN
0175-7598
URI
https://sciwatch.kiost.ac.kr/handle/2020.kiost/4495
DOI
10.1007/s00253-008-1467-5
Bibliographic Citation
APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, v.79, no.4, pp.571 - 578, 2008
Publisher
SPRINGER
Subject
THERMOSTABLE DNA-POLYMERASES; BIOCHEMICAL-CHARACTERIZATION; DEOXYRIBONUCLEIC-ACID; PYROCOCCUS-FURIOSUS; DEAMINATED BASES; HIGH-FIDELITY; URACIL; DUTPASE; CLONING; REPAIR
Keywords
dITPase; dITP generation; family B-type DNA polymerase; hypoxanthine; Thermococcus
Type
Article
Language
English
Document Type
Article
Publisher
SPRINGER
Related Researcher
Research Interests

marine biotechnology,molecular microbiology,해양생명공학,분자미생물학

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