Characterization of a dITPase from the hyperthermophilic archaeon Thermococcus onnurineus NA1 and its application in PCR amplification.
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.
Kim
Yun-Jae
YJ
Korea Ocean Research and Development Institute, Ansan, South Korea.
Ryu
Yong-Gu
YG
Lee
Hyun Sook
HS
Cho
Yona
Y
Kwon
Suk-Tae
ST
Lee
Jung-Hyun
JH
Kang
Sung Gyun
SG
D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D003854:Deoxyribonucleotides / Q000378:metabolism / Q000494:pharmacology; D007293:Inosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D007700:Kinetics; D008969:Molecular Sequence Data; D019384:Nucleic Acid Synthesis Inhibitors; D016133:Polymerase Chain Reaction / Q000187:drug effects; D011755:Pyrophosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D019712:Thermococcus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics
D019843:Archaeal Proteins; D003854:Deoxyribonucleotides; D019384:Nucleic Acid Synthesis Inhibitors; D007293:Inosine Triphosphate; D004259:DNA-Directed DNA Polymerase; D011755:Pyrophosphatases