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.
Key Laboratory of Molecular Biology on Infectious Diseases, Ministry of Education, Second Affiliated Hospital, Chongqing Medical University, Chongqing 400016, China; Department of Microbiology, College of Basic Medical Sciences, Chongqing Medical University, Chongqing 400016, China.
Zeng
Z
Tu
Key Laboratory of Molecular Biology on Infectious Diseases, Ministry of Education, Second Affiliated Hospital, Chongqing Medical University, Chongqing 400016, China.
Yuan
Y
Hu
Key Laboratory of Molecular Biology on Infectious Diseases, Ministry of Education, Second Affiliated Hospital, Chongqing Medical University, Chongqing 400016, China; Department of Microbiology, College of Basic Medical Sciences, Chongqing Medical University, Chongqing 400016, China.
Lei
L
Xu
Key Laboratory of Molecular Biology on Infectious Diseases, Ministry of Education, Second Affiliated Hospital, Chongqing Medical University, Chongqing 400016, China.
Yong
Y
Huang
Key Laboratory of Molecular Biology on Infectious Diseases, Ministry of Education, Second Affiliated Hospital, Chongqing Medical University, Chongqing 400016, China.
Xuan
X
Luo
Key Laboratory of Molecular Biology on Infectious Diseases, Ministry of Education, Second Affiliated Hospital, Chongqing Medical University, Chongqing 400016, China. Electronic address: hujieli1977@163.com.
Jie-Li
JL
Hu
Key Laboratory of Molecular Biology on Infectious Diseases, Ministry of Education, Second Affiliated Hospital, Chongqing Medical University, Chongqing 400016, China. Electronic address: ahuang1964@163.com.
Ai-Long
AL
Huang
D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D014644:Genetic Variation; D005822:Genetic Vectors; D016679:Genome, Viral / Q000235:genetics*; D006509:Hepatitis B / Q000821:virology*; D006515:Hepatitis B virus / Q000235:genetics*; D006801:Humans; D017422:Sequence Analysis, DNA
Diversity; Full-length cloning; Hepatitis B virus