Site-directed mutagenesis using Pfu DNA polymerase and T4 DNA ligase.

Here we describe a simple and cost-effective PCR-based method to introduce deletions, point mutations, frame-shift, or truncations with very high efficiency. The scope and additional applications of the method are discussed. Site-directed mutagenesis involves the introduction of mutations at the DNA level to alter the primary amino acid sequence of proteins (1,2). To approach the desired level of reliability in generating mutants, several methods have been described, some of which are technically demanding and/or require special reagents (1–6). Megaprimer-based methods for site-directed mutagenesis utilize three oligonucleotide primers (4) or only one mutagenic primer (5) in two rounds of PCR. However, when longer fragments are mutagenized, the efficiency of the methods decreases dramatically and the number of side products increases. Inverse PCR can generate mutations with two primers but solely utilizes plasmid DNA templates (6). We describe a highly efficient, cost-effective, and reliable method to generate mutations along the entire length of the cDNA. The strategy involves the following steps. (i) Four primers (e.g., Figure 1, primers #1–#4) amplify the cDNA in the first-round PCR. Primer pairs (#1 and #2) and (#3 and #4) generate the Nand C-terminal PCR products, respectively. Primers #2 and #3 are purchased as 5′ phosphorylated primers. Primers #1 and #4 are unphosphorylated. (ii) T4 DNA ligasemediated joining of the PCR products immediately following the first-round PCR. (iii) Second-round amplification of the ligated product using primers #1 and #4. (iv) Restriction digestion of the PCR product (from step 3) and cloning into any vector of choice. In our case, primer #2 contained the desired mutation (S65L, S65W, or R167Q). The PCRs contained the following components: 5.0 ng template DNA, 2.5 μL forward primers (0.05 μg/μL, 10× stock), and 2.5 μL reverse primers (0.05 μg/μL, 10× stock), 2.5 μL 2 mM dNTP, 0.5 μL 100 mM MgSO4, 2.5 μL 10× Pfu buffer (Stratagene, La Jolla, CA, USA), 0.5 μL Pfu DNA polymerase (5 U/ μL; Stratagene), and sterile water to 25 μL. Standard PCR cycling conditions were used (1 cycle of 94°C for 4 min, 54°C for 1 min, 72°C for 1 min; 28 cycles of 94°C for 1 min, 54°C for 1 min, and 72°C for 1 min; and a final extension at 72°C for 7 min). One microliter (approximately 100 ng) of each PCR product was directly used for blunt-end ligation [1 h at room temperature, 1 μL 10× buffer (New England Biolabs, Beverly, MA, USA), 1 μL each of Nand C-terminal PCR products, 0.5 μL T4 DNA ligase (100,000 U/mL), and 6.5 μL sterile water]. One microliter (approximately 10 ng) of the ligation reaction was used Site-directed mutagenesis using Pfu DNA polymerase and T4 DNA ligase