Site-Directed Mutagenesis and Gene Fusion by Megaprimer PCR

In the last few years, a variety of polymerase chain reaction (PCR)-based mutagenesis procedures have been developed (1 –16 ). Among these, the three-primer two-PCR methods (1 –3 ) represented by the original “megaprimer” technique (1 ), appear to be the simplest and most versatile ones available. A number of recent improvements and modifications of the megaprimer method have contributed to an increase in its yield, creation of a larger variety of mutations, and reduction of unwanted mutational errors (17 –23 ). The rationale of the basic method is shown schematically in Fig. 1 A , where A and B represent the “flanking” primers that can map either within the cloned gene or outside the gene (i.e., within the vector sequence) and M represents the internal “mutant” primer containing the desired base change. The first PCR is performed using the mutant primer (e.g., M1 in Fig. 1 ) and one of the flanking primers (e.g., A). The double-stranded product is purified and used as one of the primers (hence the name “megaprimer”) in the second PCR along with the other flanking primer (B). The wild-type cloned gene is used as a template in both PCR reactions. The final PCR product containing the mutation can be used in a variety of standard applications, such as cloning in expression vectors and sequencing, or in more specialized applications, such as production of the gene message in vitro if primer A (or the template sequence downstream of primer A) also contains a transcriptional promoter (e.g., that of SP6 or T7 phage). Both primers A and B are usually designed to contain convenient restriction sites so that the final, mutant PCR product can be restricted and cloned. This basic procedure can be adopted to create site-specific insertions (Fig. 1 B ), deletions (Fig. 1 C ), or gene fusions (Fig. 1 D ) by designing appropriate “mutant” primers (M) for the first PCR.