Simultaneous introduction of multiple mutations using overlap extension PCR.

Introduction of multiple mutations can be accomplished through phage M13-based site-directed mutagenesis using several oligonucleotides (6). The major drawback of this method is the low efficiency of generating all the intended mutations simultaneously (7,9). The alternative methods for introducing multisite mutations are based on polymerase chain reaction (PCR) (1) and ligase chain reaction (LCR) (8), but they require complicated modifications to the current mutagenesis and PCR protocols. Overlap extension PCR has been used to introduce single mutations embedded in the oligonucleotide primers (2) and to fuse genes or gene segments (3). We have combined both applications of the overlap extension PCR to introduce multiple mutations within genes or gene segments simultaneously. In contrast to other methods of introducing multiple mutations, the current method is fast, simple and highly efficient. In addition, this method is very flexible. To illustrate the usefulness of this method, we have applied this method to remove several restriction sites in both a promoter-modified cat gene (chloramphenicol acetyltransferase) (5) and the lacI gene. The method of Vallejo et al. (10) has been modified, and the Taq DNA polymerase has been replaced with a thermal stable polymerase with proofreading activity (Pfu). The method consists of four steps, described below. (i) The PCR primers were designed where the mutations were placed 12–13 bases upstream from the 3′ end of the cognate primers (Figure 1A). Pairs of primers annealing to the overlap regions of the mutagenic primers were used to amplify the segments of the genes that were not mutagenized. For lacI gene PCR primers (Figure 1B), the overlap regions of the mutagenic primers (B3 and B4) were shortened, and the primers, which were used to generate the unaltered segments of the gene (B2 and B5), were shifted to include some of the mutations. Primer B7 was added in the final assembly PCR to add the 3′ end. (ii) The fragments for overlap extension (Figure 1, A and B) were produced using Pfu DNA Polymerase (Stratagene, La Jolla, CA, USA) in a PerkinElmer DNA Thermal Cycler (Norwalk, CT, USA). Briefly, the plasmid carrying the modified cat gene (5) (ca. 100 ng) was mixed with 10× buffer (supplied with the enzyme), the primer pairs (25 pmol each, synthesized by GenSet, Paris, France) and dNTP (10 nmol) in a total volume of 50 μL. The mixtures were heated at 92°C for 3 min while Pfu DNA polymerase was added and cooled down to 50°C. The mixtures were incubated at 72°C for 1 min, followed by 14 cycles of PCR (92°C for 1 min; 50°C for 1 min and 72°C for 1 min) with a final extension at 72°C for 5 min. The PCR products were gelpurified (Qiagen, Hilden, Germany) and eluted in TE buffer (10 mM Tris-