title: Two-way molecular ligation for efficient conversion of monomeric hepatitis B virus DNA constructs into tandem dimers.
abstract: Replication of the 3.2-kb hepatitis B virus (HBV) genome is driven by the covalently closed circular (ccc) DNA in the nucleus, from which four classes of co-terminal RNAs are transcribed. Genome replication requires just the 3.5-kb pregenomic RNA, which is terminally redundant. Cloning the full-length HBV genome into a vector disrupts its continuity, thus preventing genome replication at the step of pregenomic RNA transcription. This can be overcome by converting the monomeric construct into a tandem dimer, yet the need to ligate two molecules of the HBV genome with vector DNA makes it inefficient and even unsuccessful. To overcome this problem we partially digested the monomeric construct with the unique restriction enzyme used for cloning, and dephosphorylated the linearized monomer before its ligation with another copy of the HBV genome. Alternatively, the monomer was linearized at another unique restriction site inside the HBV genome, followed by its dephosphorylation and ligation with another copy of the HBV genome linearized at the same site. These approaches of two-way molecular ligation greatly improved the efficiency of dimer formation with about 50% of the bacterial colonies screened harboring tandem dimers.
authors.affiliation: Department of Pathobiology and Key Laboratory of Medical Molecular Virology, School of Basic Medical Sciences, Fudan University, Shanghai, China.
authors.forename: Li
authors.initials: L
authors.lastname: Zong
authors.affiliation: Department of Infectious Diseases, Huashan Hospital, Fudan University, Shanghai, China.
authors.forename: Yanli
authors.initials: Y
authors.lastname: Qin
authors.affiliation: Department of Pathobiology and Key Laboratory of Medical Molecular Virology, School of Basic Medical Sciences, Fudan University, Shanghai, China.
authors.forename: Haodi
authors.initials: H
authors.lastname: Jia
authors.affiliation: Department of Pathobiology and Key Laboratory of Medical Molecular Virology, School of Basic Medical Sciences, Fudan University, Shanghai, China.
authors.forename: Huailiang
authors.initials: H
authors.lastname: Zhou
authors.affiliation: Department of Pathobiology and Key Laboratory of Medical Molecular Virology, School of Basic Medical Sciences, Fudan University, Shanghai, China.
authors.forename: Chaoyang
authors.initials: C
authors.lastname: Chen
authors.affiliation: Department of Pathobiology and Key Laboratory of Medical Molecular Virology, School of Basic Medical Sciences, Fudan University, Shanghai, China.
authors.forename: Ke
authors.initials: K
authors.lastname: Qiao
authors.affiliation: Department of Infectious Diseases, Huashan Hospital, Fudan University, Shanghai, China.
authors.forename: Jiming
authors.initials: J
authors.lastname: Zhang
authors.affiliation: Department of Pathobiology and Key Laboratory of Medical Molecular Virology, School of Basic Medical Sciences, Fudan University, Shanghai, China.
authors.forename: Yongxiang
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Liver Research Center, Rhode Island Hospital, Brown University, Providence, RI, USA.
authors.forename: Jisu
authors.initials: J
authors.lastname: Li
authors.affiliation: Department of Pathobiology and Key Laboratory of Medical Molecular Virology, School of Basic Medical Sciences, Fudan University, Shanghai, China; Liver Research Center, Rhode Island Hospital, Brown University, Providence, RI, USA. Electronic address: shupingtong@fudan.edu.cn.
authors.forename: Shuping
authors.initials: S
authors.lastname: Tong
mesh_terms: D003001:Cloning, Molecular; D004270:DNA, Circular; D004279:DNA, Viral; D005822:Genetic Vectors; D016679:Genome, Viral; D006515:Hepatitis B virus / Q000235:genetics*; D006801:Humans; D010957:Plasmids / Q000235:genetics
chemical_list: D004270:DNA, Circular; D004279:DNA, Viral
keywords: Covalently closed circular DNA; Hepatitis B virus; Pregenomic RNA; Replication; Tandem dimer