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.
Crisci S，Amitrano F，Saggese M，et al.Overview of Current Targeted Anti-Cancer Drugs for Therapy in Onco-Hematology[J].Medicina（Kaunas），2019，55（8）：414.
[9]
Qu G，Li A，Acevedo-Rocha CG，et al.The Crucial Role of Methodology Development in Directed Evolution of Selective Enzymes[J].Angew Chem Int Ed Engl，2020，59（32）：13 204-13 231.
[10]
Sayous V，Lubrano P，Li Y，et al.Unbiased libraries in protein directed evolution[J].Biochim Biophys Acta Proteins Proteom，2020，1868（2）：140 321.
[11]
She W，Ni J，Shui K，et al.Rapid and Error-Free Site-Directed Mutagenesis by a PCR-Free In Vitro CRISPR/Cas9-Mediated Mutagenic System[J].ACS Synth Biol，2018，7（9）：2236-2244.
[12]
Kanazashi Y，Hirose A，Takahashi I，et al.Simultaneous site-directed mutagenesis of duplicated loci in soybean using a single guide RNA[J].Plant Cell Rep，2018，37（3）：553-563.
[13]
WatanabeK，TsuchidaY，OkibeN，etal.ScanningtheCorynebacterium glutamicum R genome for high-efficiency secretion signal sequences[J].Microbiology，2009，155（Pt 3）：741-750.
[14]
Wang X，Mathieu M，Brezski RJ.IgG Fc engineering to modulate antibody effector functions[J].Protein Cell，2018，9（1）：63-73.
[15]
Wardemann H，Busse CE.Expression Cloning of Antibodies from Single Human B Cells[J].Methods Mol Biol，2019，1956：105-125.
京公网安备 11010502046607号