中国生物工程杂志

Objectives: To introduce site-directed mutagenesis into the pcDNA3.1(+)-F plasmid containing respiratory syncytial virus F gene coding sequence by single circular PCR using single primer. Methods: First, three single-stranded primers with mutagenesis N70Q, I431N or Q270T were designed according to the template pcDNA3.1(+)-F plasmid, respectively. Second, once-single PCR with the double-stranded DNA template and each of the three single primers was performed. Next, the PCR products were treated with endonuclease Dpn I to eliminate the methylated template DNA and then transformed into E. coli DH5α. Finally, plasmids were extracted from the culture of the selected positive clones and sent for sequencing after digestion identification. Results: The results of enzymatic digestion and sequencing analysis were as expected. Three types of site-directed mutagenesis were successfully introduced, namely ‘_X_’, ‘X_X’, ‘XXX’ when ‘X’ means mutated nucleotide and ‘_’ means the opposite. Conclusions: Single-primer PCR is a simple, quick and effective innovation to introduce site-directed mutagenesis, which solved the problems of multiple PCRs, complicated procedures and low effectiveness in previous methods.