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.
Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
