Objective To explore the optimal primer ratio and concentration of asymmetric polymerase chain reaction(A-PCR)in producing hepatitis B virus(HBV)single-stranded DNA(ssDNA)for pyrosequencing.Methods A-PCR was carried o...Objective To explore the optimal primer ratio and concentration of asymmetric polymerase chain reaction(A-PCR)in producing hepatitis B virus(HBV)single-stranded DNA(ssDNA)for pyrosequencing.Methods A-PCR was carried out to generate HBV ssDNA with forward to reverse primers of different ratios(50∶1,100∶1)and concentrations(13.0 pmol/25μL and 0.14 pmol/25μL,19.5 pmol/25μL and 0.21 pmol/25μL),and the product yield and quality were compared respectively.Results The forward to reverse primer ratio of 50∶1 provided better yield and concentration of 19.5 pmol/25μL and 0.21 pmol//25μL generated a clearer band.Conclusion A simple and feasible method to produce HBV ssDNA for pyrosequencing in batch is established.展开更多
基金supported by the National Natural Science Foundation of China(No.60878056)the Doctoral Foundation of Xi’an Jiaotong University(DFXJTU2004-12)
文摘Objective To explore the optimal primer ratio and concentration of asymmetric polymerase chain reaction(A-PCR)in producing hepatitis B virus(HBV)single-stranded DNA(ssDNA)for pyrosequencing.Methods A-PCR was carried out to generate HBV ssDNA with forward to reverse primers of different ratios(50∶1,100∶1)and concentrations(13.0 pmol/25μL and 0.14 pmol/25μL,19.5 pmol/25μL and 0.21 pmol/25μL),and the product yield and quality were compared respectively.Results The forward to reverse primer ratio of 50∶1 provided better yield and concentration of 19.5 pmol/25μL and 0.21 pmol//25μL generated a clearer band.Conclusion A simple and feasible method to produce HBV ssDNA for pyrosequencing in batch is established.
