Megaprimer PCR: Application in Mutagenesis and Gene Fusion

Since the advent of the polymerase chain reaction (PCR), a variety of PCR-based procedures of mutagenesis have been developed through the use of synthetic primers encoding the mutation. Among these, the megaprimer method and related ones (1 –5 ) remain some of the simplest and most versatile. Variations and improvements of the basic technique have been suggested over the past few years; these include a combination of magapriming and overlap extension, improvement of yield, use of single-stranded DNA, avoidance of unwanted mutations arising from nontemplated insertions by Taq polymerase, and the inclusion of various kinds of mutations, including multiple, nonadjacent ones (2 –11 ). The basic method (Fig. 1 ) requires three oligonucleotide primers and two PCRs (termed PCR-1 and -2 here) using the wild-type DNA as template (1 ,2 ,8 ,10 ). The “mutant” primer is represented by M and the two “outside” primers by A and B. The M primer may encode a substitution, a deletion, an insertion, or a combination of these mutations, thus providing versatility while using the same basic strategy (10 ). The frist PCR (PCR-1) is performed using the mutant primer M and one of the outside primers, such as A (Fig. 1 ). The double-stranded product A-M is purified and used as a primer (hence the name megaprimer; ref. 1 ) in the second PCR (PCR-2) together with the other outside primer, B. Although both strands of the megaprimer may prime on the respective complementary strands of the template, the fundamental principles of PCR amplification ensure that only the one that extends to the other primer, that is, B in Fig. 1 , will be exponentially amplified into the double stranded product in PCR-2.