Site-directed mutagenesis for large plasmids is a difficult task that cannot easily be solved by the conventional methods used in many laboratories. In this study, we developed an effective method for Site-directed Mutagenesis for Large Plasmids (SMLP) based on a PCR technique. The SMLP method combines several effective approaches, including a high-efficiency DNA polymerase for the large DNA amplification, two independent PCR reactions and a fast recombinational ligation. Using this method, we have achieved a variety of mutants for the filamin A gene (7.9 kb) cloned in the pcDNA (5.4 kb) or the pLV-U6-CMV-EGFP (9.4 kb) plasmids, indicating that this method can be applied to site-directed mutagenesis for the plasmids up to 17.3 kb.
Fig: A summary showing the methods established for PCR-based site-directed mutagenesis. (A) A diagram for site-directed mutagenesis based on PCR, overlap extension PCR, and gene cloning. In this method, two PCR reactions are first performed with the primer pair of CFP and MRP and the primer pair of MFP and CRP. PCR products are purified and the resulting PCR products were used for overlap extension PCR with the primer pair of CFP and CRP. The products from overlap extension PCR were digested with restriction enzymes, and the resulting fragments were ligated with the plasmids digested with the same restriction enzymes. CFP, cloning forward primer; CRP, cloning reverse primer; MFP, mutation forward primer; MRP, mutation reverse primer. The red stars in the left and right panels represent mutated bases. (B) A diagram for site-directed mutagenesis based on PCR with a pair of complementary primers (double-strand DNA fragments), Dpn I digestion, and transformation. (C) A diagram for site-directed mutagenesis based on PCR with a pair of partially complementary primers, Dpn I digestion and transformation (a) or based on PCR with a pair of partially complementary DNA fragments, recombinational ligation, and transformation (b). (D) A diagram for site-directed mutagenesis based on PCR with a pair of inverse primers, phosphorylation, ligation, and transformation. The blue dots in the left panel represent phosphorylated groups. The red stars in A-D represent mutated bases.
We show that the SMLP method has a greater advantage than the conventional methods tested in this study, and this method can be applied to substitution, deletion, and insertion mutations for both large and small plasmids as well as the assembly of three fragments from PCR reactions. Altogether, the SMLP method is simple, effective, and beneficial to the laboratories that require completing the mutagenesis of large plasmids.
Zhang, K., Yin, X., Shi, K. et al. A high-efficiency method for site-directed mutagenesis of large plasmids based on large DNA fragment amplification and recombinational ligation. Sci Rep 11,10454 (2021). https://doi.org/10.1038/s41598-021-89884-z