An improved method for simple and efficient hepatitis B virus genome cloning.

The genetic variation of hepatitis B virus (HBV) is associated with a natural history of infection and with a response to antiviral therapy. Current attempts to investigate the genetic variation within patients infected with HBV have characterized only the subgenomic region or a limited number of full-length genomes. In this study, a simple and efficient full-length HBV cloning method, which is independent of restriction digestion and ligation, from a clinical sample is presented. The full-length HBV amplified from patients' sera serves as two megaprimers and contains two ends that are homologous to the insertion site of a newly constructed plasmid. This method could enable the extension of the plasmid backbone by DNA polymerase. To improve the cloning efficiency, a LacZ gene fragment was incorporated into the middle of the insertion site in the plasmid and was used for blue-white screening. Through optimization and evaluation, a high success rate of positive clones (90%) from an individual patient was obtained. Thus, this study provides a simple, efficient, and convenient method for full-length HBV cloning from many clinical samples, and this method could greatly facilitate studies concerning the genetic variation of HBV.