Megaprimer mutagenesis using very long primers.

It is well known that some genetic mutations have a critical impact on protein structure and function. To study the effects of these mutations, site-directed mutagenesis allows the introduction of a defined substitution of specific residues. For the past few years, numerous PCR-based methods have been employed for achieving site-directed mutagenesis. Among these methods, megaprimer PCR has been widely employed because of its simplicity and low cost (5). In brief, megaprimer PCR utilizes three PCR primers in two rounds of PCR amplification. In the first round of PCR amplification, the external forward primers (F) and internal mutant reverse primers (M) were responsible for inducing a specific mutation into the PCR product. In the second round of PCR amplification, the PCR products from the first round of PCR amplification act as the megaprimers, together with the external reverse primers (R), and the full length of the target sequence is amplified with the desired mutation. Based on this principle, numerous different approaches have emerged to achieve accurate induction of mutations (2,3,6). These studies relied on megaprimers that were around 300 bp. Other studies have indicated that, for megaprimers longer than 300 bp, an excess amount of template is required to produce the desired PCR product yield (1). However, in our hands, the use of these methods (1,2,6) with a much longer megaprimer (800 bp in length) resulted in a very low percentage of clones that carried a desired mutation. Here we report the development of a simple and reliable adapted method for longer megaprimer site-directed mutagenesis. Our new site-directed mutagenesis strategy has been applied successfully to generate several mutants of the tumor suppressor gene, p53, and mutants in Kras. The method successfully generated several “hot spot” mutations including the p53 codon 282 mutation. To illustrate the method, only the codon 282 results will be presented here. Figure 1 outlines the overall procedure. The method consists of two PCR amplifications. In the first round of PCR, the reaction was carried out in a total volume of 50 μL. The reaction consisted of 2.5 U PfuTurbo® DNA polymerase (Stratagene, La Jolla, CA, USA), PCR buffer [20 mM Tris-HCl, 2 mM MgSO4, 10 mM KCl, 10 mM (NH4)2SO4, 0.1% Triton® X-100, and 0.1% BSA], 320 pg/μL wild-type p53 inserted in pcDNA vector (Promega, Madison, WI, USA), 0.4 mM dNTP, 0.2 mM F primer (5′-GCCTAAGCTTTCACTGCCATGGAGGAG-3′) and 0.2 mM M primer (5′-CTTCCTCTGTGCGCCAGTCTCTCC-3′) (Figure 1). First-round PCR was performed under the following conditions: 95°C for 5 min of initial denaturation, followed by 20 cycles of template denaturation at 94°C for 1 min, primer annealing at 55°C for 1 min, and primer extension at 72°C for 1.5 min. The reaction was finished with final extension at 72°C for 10 min. An important modification of previous methods is that, after the first round of PCR amplification, only primers were removed by using PCR purification (Qiagen, Valencia, CA, USA). In the second round of PCR amplification, the reaction was also carried out in 50 μL reaction volume. It consisted of 10 μL “carryover” first-round PCR product, which was approximately 6 ng/μL, 2.5 U PfuTurbo DNA polymerase, PCR buffer, 0.4 mM dNTP, and 2 mM MgCl2. Because of the low full-length template concentration, the mixture was initially heated at 95°C (for 5 min for initial denaturation) and followed by five cycles of asymmetric PCR, where the templates were denatured at 95°C for 1 min and only megaprimer annealed and extended at 72°C for 3 min. According to two previous studies (3,7), this asymmetric PCR will increase the product yield when limited templates were available at the start of the PCR cycle. At the end of asymmetric PCR, R primer (5′-GAAGTCTAGAATGTCAGTCTGAGTCAGG-3′) (Figure 1) was added into the reaction mixture to 0.2 mM. The full length of p53 mutant cDNA was amplified by 20 PCR cycles, as described for the first-round PCR. Finally, the PCR products were purified and digested with HindIII and XbaI restriction enzymes (New England Biolabs, Beverly, MA, USA) and cloned Benchmarks