Asymmetric PCR method in generation of HBV ssDNA for pyrosequencing / 西安交通大学学报·英文版

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.