title: Parallel assembly for multiple site-directed mutagenesis of plasmids.
abstract: A parallel assembly method for multiple site-directed mutagenesis of plasmids was developed here based on Golden Gate cloning. It takes advantage of type IIs restriction enzymes and T4 DNA ligase to assemble multiple DNA fragments into a plasmid by a defined order. This method can accommodate multiple plasmid mutagenesis at any desired position with all three sequence modification types (substitution, deletion, and insertion) simultaneously. Furthermore, it can be used to create otherwise difficult-to-make mutants-larger deletions and insertions and mutagenesis on larger plasmids. The processes of mutagenesis can be completed quickly by a single restriction-ligation reaction.
authors.affiliation: Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Science, Haikou, China.
authors.forename: Pu
authors.initials: P
authors.lastname: Yan
authors.forename: XinZheng
authors.initials: X
authors.lastname: Gao
authors.forename: Wentao
authors.initials: W
authors.lastname: Shen
authors.forename: Peng
authors.initials: P
authors.lastname: Zhou
authors.forename: Jun
authors.initials: J
authors.lastname: Duan
mesh_terms: D017122:Bacteriophage T4 / Q000201:enzymology; D011088:DNA Ligases / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D010957:Plasmids / Q000235:genetics* / Q000378:metabolism; D016133:Polymerase Chain Reaction
chemical_list: D015252:Deoxyribonucleases, Type II Site-Specific; D011088:DNA Ligases