Modification of a PCR-based site-directed mutagenesis method.

Site-directed mutagenesis is a powerful tool for producing mutants to assess the importance of specific amino acid residues in a protein’s structure and/or function. We wanted to generate mutants of human ETS1 cDNA in the pET15b vector (Novagen, Madison, WI, USA) from which we had been producing wild-type protein for structural studies (11). Since we were subject to the constraints of this vector, we looked to established polymerase chain reaction (PCR)-based methods for generating site-directed mutants (1–5,9, 10,12). However, these methods were labor-intensive (1,5), did not yield the expected mutagenesis efficiency (4,10), required unavailable restriction enzyme sites (9) or necessitated introduction of restriction sites (3,12) that would have altered additional residues in the coding sequence. As a result, we modified several aspects of the method of Weiner et al. (10) to create a PCR-based site-directed mutagenesis method with higher mutagenesis efficiency that can be used to mutate any site within a protein's coding sequence without additional changes. This method can be used with any vector, uses only two specific primers in a single PCR and yields mutant-encoding colonies in less than 48 h with an overall efficiency of 80%. As mentioned above, a similar sitedirected mutagenesis protocol has been published by Weiner et al. (10), but in the method described here, several steps have been modified or eliminated, resulting in a simplified procedure and a higher mutation efficiency. In particular, our procedure uses Pfu DNA polymerase, which not only increases the fidelity of PCR over that performed with Taq DNA polymerase, but also generates blunt-ended products, eliminating the need for end polishing (6,8). As in the Weiner et al. protocol (10), elimination of wild-type template DNA is accomplished by DpnI digestion, which is specific for methylated substrates (7). Since template plasmids isolated from most common laboratory bacterial strains are methylated, these templates would be recognized and digested by DpnI, whereas the unmethylated PCRamplified DNA would remain intact. In an experiment in which the reaction was not subjected to DpnI digestion, the mutagenesis efficiency dropped from 80% to 40% (data not shown), demonstrating the necessity of DpnI digestion for high mutagenesis efficiency. In contrast to the Weiner et al. procedure (10), ligation precedes DpnI digestion, eliminating the creation of religated, partially digested parental template products and increasing the mutagenesis efficiency. In addition, the low amount of template used in the method described here also increases the mutagenesis efficiency. An overview of this site-directed mutagenesis protocol is shown in Figure 1. Adjacent primers were designed on opposite DNA strands with the mutation encoded at the 5′ end of one primer. Both primers were phosphorylated before PCR by suspension of 50 pmol/μL of primer in ligase buffer (50 mM Tris-HCl, pH 7.6, 10 mM MgCl2, 1 mM ATP, 1 mM dithiothreitol [DTT], 5% polyethylene glycol [PEG]-8000) with 1 U/μL T4 polynucleotide kinase (Amersham, Arlington Heights, IL, USA), followed by a 30-min incubation at 37°C. PCR was carried out in a 50-μL mixture using 1.25 U Pfu DNA Polymerase (Stratagene, La Jolla, CA, USA) with 10–50 ng template plasmid DNA, 50 pmol of each phosphorylated primer, 200 μL each dNTP in supplied Pfu reaction buffer [20 mM Tris-HCl, pH 8.8, 10 mM KCl, 10 mM (NH4)2SO4, 2 mM MgSO4, 0.1% Triton X-100, 100 μg/mL bovine serum albumin (BSA)]. Amplification parameters were an initial 4 min at 94°C, followed by 16 cycles of 1 min at 94°C, 1 min at the annealing temperature [10° below the calculated primer melting temperature (4× G/C + 2× A/T)] and 15 min (2.5 min/kb) plus 5 s auto-extension at 72°C. After PCR, 5 μL of the reaction mixture were run on a 1% agarose gel to confirm amplified product formation. PCR samples were then phenol/chloroform-extracted and ethanolprecipitated. Samples were resuspended in 20 μL ligase buffer with 10 U of T4 DNA ligase (Life Technologies,