Enhanced Mutant Screening in One-step PCR-based Multiple Site-directed Plasmid Mutagenesis by Introduction of Silent Restriction Sites for Structural and Functional Study of Proteins

Biological Procedures Online

Short Report

Ting-Yu Kuo¹  Chung-Che Tsai¹  Hua-Wen Fu² 

[1] Institute of Molecular and Cellular Biology, National Tsing Hua University, 30013, Hsinchu, Taiwan, Republic of China;Institute of Molecular and Cellular Biology, National Tsing Hua University, 30013, Hsinchu, Taiwan, Republic of China;Department of Life Science, National Tsing Hua University, 30013, Hsinchu, Taiwan, Republic of China;

关键词: Site-directed mutagenesis;    Polymerase chain reaction;    PCR;    Silent restriction mutation;    Helicobacter pylori;   

DOI  :  10.1186/s12575-017-0062-5

 received in 2017-06-22, accepted in 2017-09-19,  发布年份 2017

来源: Springer

【 摘 要 】

Site-directed mutagenesis (SDM) has been widely used for studying the structure and function of proteins. A one-step polymerase chain reaction (PCR)-based multiple site-directed plasmid mutagenesis method with extended non-overlapping sequence at the 3′ end of the primer increases the PCR amplification efficiency and the capacity of multi-site mutagenesis. Here, we introduced silent restriction sites in the primers used in this PCR-based SDM method by utilizing SDM-Assist software to generate mutants of Helicobacter pylori neutrophil-activating protein (HP-NAP), whose gene has low GC content. The HP-NAP mutants were efficiently generated by this modified mutagenesis method and quickly identified by a simple restriction digest due to the presence of the silent restriction site. This modified PCR-based SDM method with the introduction of a silent restriction site on the primer is efficient for generation and identification of mutations in the gene of interest.

【 授权许可】

CC BY   
© The Author(s). 2017