PCR-directed in vitro mutagenesis using a 'temporary' restriction site

Abstract

▼PCR mutagenesis close to the end of a target sequence or close to a natural restriction site anywhere within the target sequence can be performed with a single PCR reaction, using mismatching primers that also include the neighboring restriction site (Ref. 1). If natural restriction sites are not available near the site where a point mutation, deletion, insertion, or fusion to another sequence is planned, different strategies are possible. The segments upstream and downstream of the mutation site or the fusion site can be amplified using overlapping primers in separate PCR reactions. The two products are then joined together in a third PCR reaction, based on overlap extension (Ref. 2). This method requires long overlapping primers, and the overlap extension reaction is often difficult or unsuccessful, either because the overlap is too short or because of adverse secondary structures. Another technique for mutations within a single sequence is to use the product of a first mutagenic PCR reaction as a megaprimer to amplify the entire sequence (Ref. 3). Alternatively, a suitable restriction site can be introduced by silent mutations that are close to the primary site of mutagenesis or to the desired site of fusion of two sequences. Again, this requires two separate PCR reactions to amplify the upstream and the downstream segments with the new restriction site at the ends. At this site, the products can simply be ligated. However, it is often difficult or impossible to find a suitable unique restriction sequence that can be generated by silent mutations within a short distance of a given position. Here, we describe a simple strategy to introduce a temporary restriction site near the place of mutation, such that the upstream and the downstream PCR products can be fused readily by ligation of their ‘sticky ends’, after which they can easily be removed. The principle is to insert four additional nucleotides into the sequence that, together with the flanking nucleotides at the insertion point, constitute