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Parallel assembly for multiple site-directed mutagenesis of plasmids
Authors:Pu Yan  Xinzheng Gao  Wentao Shen  Peng Zhou  Jun Duan
Affiliation:Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Science, Haikou 571101, China; South China Botanical Garden, Chinese Academy of Sciences, Guangzhou 510650, China.
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.
Keywords:
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