| Abstract: | 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. |
