Mutagenesis and Gene Fusion by Megaprimer PCR

Among the many variations of polymerase chain reaction (PCR)-based mutagenesis procedures, the “megaprimer” method (1 –5 ) is probably the simplest and most versatile. The method utilizes three oligonucleotide primers and two rounds of PCR performed on a DNA template containing the cloned gene which is to be mutated. The rationale of the basic method is shown schematically in Fig. 1A , 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 round of 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 round of PCR along with the other flanking primer (B). The wild-type cloned gene is used as 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). This basic procedure can be adopted to create site-specific insertions (Fig. 1B ), deletions (Fig. 1C ), and gene fusions (Fig. 1D ). Successful and error-free PCR in the second round often requires special considerations. The reader is, therefore, strongly urged to go through the whole chapter, including Section 4. , before proceeding with the actual experiment.

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