We constructed hepatocellular carcinoma (HCC) cells that stably express stathmin with a Ser25 phosphorylation site mutation (stathmin S25A). We used the polymerase chain reaction for site-directed mutagenesis, constructed a stathmin S25A plasmid, and verified the results by restriction enzyme cleavage and sequencing technology. Using the liposome transfection method, stathmin wild-type and S25A HCCLM6 cells were established, which were identified by western blotting.
Gene mutation plays an important role in molecular biological studies. A highly efficient one-step polymerase chain reaction-based mutagenesis technique for site-directed mutagenesis was developed in this study. One complementary pair of primers was designed that contained the desired mutations in the middle of the primers. The amplification products of mutation were amplified using a high-fidelity DNA polymerase and the original plasmid templates were digested by DpnI.
Site-directed mutagenesis is an essential technique for investigating the mechanisms of gene regulation on a molecular level, as well as for exploring post-translational modifications and functional structure at the protein level. Polymerase chain reaction combining in vitro synthesis of oligonucleotide primers allows for site-directed mutation to be performed with ease. However, site-directed mutagenesis is difficult when larger plasmids are involved.