期刊
ANALYTICAL BIOCHEMISTRY
卷 430, 期 1, 页码 65-67出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ab.2012.07.029
关键词
Multiple mutagenesis; Plasmid; Golden Gate cloning; Parallel assembly
资金
- National Natural Science Foundation of China [30960218]
- National Nonprofit Institute research grant of Institute of Chinese Academy of Tropical Agricultural Science-Tropical Bioscience and Biotechnology (CATAS-ITBB [ITBB110212]
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. (c) 2012 Elsevier Inc. All rights reserved.
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