title: Single primer-mediated circular polymerase chain reaction for hairpin DNA cloning and plasmid editing.
abstract: We developed and validated a universal polymerase chain reaction (PCR) method, single primer circular (SPC)-PCR, using single primer to simultaneously insert and amplify a short hairpin sequence into a vector with a high success rate. In this method, the hairpin structure is divided into two parts and fused into a vector by PCR. Then, a single primer is used to cyclize the chimera into a mature short hairpin RNA (shRNA) expression vector. It is not biased by loop length or palindromic structures. Six hairpin DNAs with short 4-nucleotide loops were successfully cloned. Moreover, SPC-PCR was also applied to plasmid editing within 3 h with a success rate higher than 95%.
authors.affiliation: Lab of Molecular Immunology, State Key Lab of Genetic Engineering, Department of Microbiology, Institute of Biomedical Sciences, School of Life Science, Fudan University, Shanghai 200433, China; Department of Clinical Laboratory, Lishui Central Hospital of Zhejiang University, Zhejiang 323000, China.
authors.forename: Jiansheng
authors.initials: J
authors.lastname: Huang
authors.affiliation: Lab of Molecular Immunology, State Key Lab of Genetic Engineering, Department of Microbiology, Institute of Biomedical Sciences, School of Life Science, Fudan University, Shanghai 200433, China.
authors.forename: Inamullah
authors.initials: I
authors.lastname: Khan
authors.affiliation: Lab of Molecular Immunology, State Key Lab of Genetic Engineering, Department of Microbiology, Institute of Biomedical Sciences, School of Life Science, Fudan University, Shanghai 200433, China.
authors.forename: Rui
authors.initials: R
authors.lastname: Liu
authors.affiliation: Lab of Molecular Immunology, State Key Lab of Genetic Engineering, Department of Microbiology, Institute of Biomedical Sciences, School of Life Science, Fudan University, Shanghai 200433, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Yang
authors.affiliation: Lab of Molecular Immunology, State Key Lab of Genetic Engineering, Department of Microbiology, Institute of Biomedical Sciences, School of Life Science, Fudan University, Shanghai 200433, China. Electronic address: nzhu@fudan.edu.cn.
authors.forename: Naishuo
authors.initials: N
authors.lastname: Zhu
mesh_terms: D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics*; D017931:DNA Primers; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods*
chemical_list: D017931:DNA Primers; D004247:DNA
keywords: Hairpin structure cloning; Plasmid editing; SPC–PCR; Single primer