![]() The red stars in the left and right panels represent mutated bases. CFP, cloning forward primer CRP, cloning reverse primer MFP, mutation forward primer MRP, mutation reverse primer. 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. PCR products are purified and the resulting PCR products were used for overlap extension PCR with the primer pair of CFP and CRP. 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. ( A) A diagram for site-directed mutagenesis based on PCR, overlap extension PCR, and gene cloning. On this occasion, the gene mutants cannot be obtained by directly transforming PCR products into bacteria after Dpn I digestion unless the PCR products are purified and subjected to recombinational ligation and transformation.Ī summary showing the methods established for PCR-based site-directed mutagenesis. Although the partially complementary primers reduce the chance to form the primer dimers during PCR, PCR products with homologous ends are possibly produced in the reaction (see the summary in Fig. To overcome this drawback, scientists have developed an improved method using a pair of partially complementary primers in PCR reaction 6, 11. However, the efficiency of PCR reaction in this method is extremely low due to the formation of primer-dimers, which often leads to the failure of mutagenesis. For instance, the Quick-Change Site-Directed Mutagenesis Kit (Thermo Scientific), one of the site-directed mutagenesis kits mostly used, has been developed based on this method. This method has widely been applied to the generation of gene mutants in many laboratories. One of the simplest methods for site-directed mutagenesis is the method based on PCR with a pair of complementary primers (a double-stranded DNA fragment), where the plasmids acting as DNA templates during PCR are required to be digested by the restriction enzyme DpnI before transformation 9, 10. However, the drawbacks of this method are that the overlap extension reaction fails frequently, and the method is labour-consuming. The products from the overlap extension PCR reaction are eventually cloned into plasmids. In this method two independent PCR reactions are performed, the resulting PCR products are used for an overlap extension PCR reaction. So far, many PCR-based methods for site-directed mutagenesis have been developed 5, 6 among which, site-directed mutagenesis by overlap extension PCR has first been developed 7, 8. Although gene mutations, including point substitution, deletion, and insertion, can be achieved by a variety of methods 5, 6 site-directed mutagenesis mediated by polymerase chain reaction (PCR) is one of the most powerful approaches to generate gene mutants in vitro. Site-directed mutagenesis, one of the most important techniques in molecular biology, has widely been used to investigate the structures and functions of nucleic acids and proteins, the mechanisms of genetic diseases, and the effect of genome modification 1, 2, 3, 4. Altogether, the SMLP method is simple, effective, and beneficial to the laboratories that require completing the mutagenesis of large plasmids. 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. 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. 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. In this study, we developed an effective method for Site-directed Mutagenesis for Large Plasmids (SMLP) based on a PCR technique. Site-directed mutagenesis for large plasmids is a difficult task that cannot easily be solved by the conventional methods used in many laboratories.
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