Optimized inverse PCR strategy for constructing multilocus mutants efficiently.

Bilin XU; Qing Zhu; Yanyan Chen; Yongliang Zheng

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Optimized inverse PCR strategy for constructing multilocus mutants efficiently.

Author: Bilin XU ¹ ; Qing ZHU ² ; Yanyan CHEN ¹ ; Yongliang ZHENG ¹
Author Information

1. Hubei Key Laboratory of Economic Forest Germplasm Improvement and Resources Comprehensive Utilization, Hubei Collaborative Innovation Center for the Characteristic Resources Exploitation of Dabie Mountains, College of Biological and Agricultural Resources, Huanggang Normal University, Huanggang 438000, Hubei, China.
2. Wuhan Center for Disease Control and Prevention, Wuhan 430015, Hubei, China.
Publication Type:Journal Article
Keywords: multilocus mutant; primer; reverse PCR; site-directed mutagenesis
MeSH: DNA Primers; genetics; Escherichia coli; Mutagenesis, Site-Directed; methods; Plasmids; genetics; Polymerase Chain Reaction
From: Chinese Journal of Biotechnology 2020;36(4):801-809
CountryChina
Language:Chinese
Abstract: Mutants of proteins are the basis for studying their structure and function, this work aimed to establish an efficient and rapid method for constructing multi-site mutants. When four or more adjacent amino acid residues need to be mutated, firstly, two long and two short primers (long primers Ⅰ/Ⅰ, short primersⅡ/Ⅱ) were designed: the long primers contain mutated sites, and the number of mutant bases is ≤20 bp, the short primers do not contain mutated sites; GC contents of the long and short primers are ≤80%, and the difference of annealing temperature is ≤40 °C. Then two sets of reverse PCR amplifications were performed using primer pairs (Ⅰ/Ⅱand Ⅰ/Ⅱ) and templates, respectively. After amplification, each system can obtain non-methylated linear plasmids which contain mutated sites, and the breakpoints of the two sets of linear plasmids amplified by primers Ⅰ/Ⅱ and Ⅲ/Ⅳ were distributed on both sides of the mutated sites. Followed by digested by DpnⅠ to remove the methylated templates, the recovered PCR products, which were mixed in an equimolar ratio, were performed another round of denaturation and annealing: the two sets of linear plasmids were denatured at 95 °C and then annealed with each other's single-stranded DNA as templates to form open-loop plasmids, and then the transformants containing the mutations will be obtained after transformed the open-loop plasmids into Escherichia coli competent cells. Results showed that, this method can mutate 4 to 11 consecutive amino acid residues (8-20 bp) simultaneously, which will greatly simplify the construction of multi-site mutants, Thereby improve the efficiency of protein structure and function research further.