The PCR is of particular use in the construction of precisely engineered DNA molecules for applications such as site-directed mutagenesis or gene recombinations. One important class of mutational variants are those with large deletions which are often difficult to generate by traditional mutagenesis techniques. Such deletions can be generated using the PCR overlap extension procedure (Higuchi et al., 1988; Horton and Pease, 1991) which requires two mutagenic primers. An alternative one-sided (megaprimer) procedure uses only one mutagenic primer but results in lower mutant yield (Horton and Pease, 1991) and is of limited use in constructing deletions. Here we describe a megaprimer approach for generating deletions with high efficiency by a two step PCR procedure. A restricted fragment of DNA is used as a template in the second PCR and offers several advantages which are described below. This approach was used to delete a 460 bp region of the gao A gene encoding the N-terminal domain of galactose oxidase of Dactylium dendroides (Ito et al., 1991; McPherson et al., 1992).

The overall procedure (Figure 1A) involves two PCR amplifications which use the mutagenic primer and two short flanking primers that may be either gene-or vector-specific primers depending on availability and subsequent cloning strategy. In the first PCR (PCR1), the deletion primer (d; Figure IB) and upstream primer (a) are used to amplify a DNA fragment (megaprimer; Sarkar and Sommers, 1990) which may be either double or single stranded if produced by asymmetric PCR (Perrin and Gilliland, 1990). The 3'end of this megaprimer derived from the 5'half of the deletion primer (see Figure 1A and B) is complementary to the downstream deletion end-point. In the second PCR (PCR2), primers a and b and the megaprimer are used in the presence of a wild-type DNA fragment which, importantly, lacks the sequence corresponding to the template region amplified during PCR1. During the initial cycles of PCR2 the megaprimer, primer b and primer a sequentially synthesize the mutant fragment (see Figure 1A) which is then amplified by primers a and b in the subsequent cycles of the PCR. The second strand of the megaprimer fragment is non-productive in PCR2.