环状定点诱变技术在载体构建中的应用

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Abstract Objective To explore the application of circular site-directed mutagenesis technology in single base mutation, restriction site modification and fragment deletion of the target plasmid. Methods The primer pairs were designed including target mutation, partial complementation, and 5’ end contains 5-8 bp protruding. The circular plasmid was used as template for amplification and digested and removed by methylase enzyme DpnI. Transform DH5α for gap repair then site-directed mutation plasmid was obtained. Results Through the validation by enzyme cutting and gene sequencing, circular site-directed mutagenesis technology could be efficiently used in single base mutation, modification of enzyme site and deletion of fragment. Conclusion The circular site-directed mutagenesis technology is simple, efficient and quick to realize single base mutation, restriction site modification and fragment deletion of the target plasmid,which can basically meet the needs of molecular construction in the process of drug development.

Key words： Circular site-directed mutagenesis technology Fragment deletion Restriction enzyme cutting site PCR amplification

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	Articles by authors
	WANG Bin
	XING Ti-kun
	SONG Lu-ping
	YANG Zhen-ping
	JING Xin-rui
	WANG Ya-ping
	HUANG Shuai
	ZHANG Jing-jing

Cite this article:

WANG Bin,XING Ti-kun,SONG Lu-ping, et al. Application of circular site-directed mutagenesis in vector construction[J]. 中国当代医药, 2020, 27(26): 32-34转38.

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https://www.dangdaiyiyao.com/EN/ OR https://www.dangdaiyiyao.com/EN/Y2020/V27/I26/32

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