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 N- and C-terminal PCR products, respectively. Primers #2 and #3 are purchased as 5′ phosphorylated primers. Primers #1 and #4 are unphosphorylated. (ii) T4 DNA ligase-mediated 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 micro-liter (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 N- and C-terminal PCR products, 0.5 μL T4 DNA ligase (100,000 U/mL), and 6.5 μL sterile water]. One micro-liter (approximately 10 ng) of the ligation reaction was used for the second-round PCR under the conditions described above. Final PCR products were digested with restriction enzymes and gel-purified using the Zymoclean Gel RNA Recovery Kit (Zymo Research, Orange, CA, USA). Fifty nanograms of insert were ligated to compatible sites in 50 ng mammalian expression vector, pEGFP-C1, and transformed into chemically competent bacteria. Open in a separate window Figure 1 Strategy for site-directed mutagenesis Step 1. Introduction of a deletion shown as an example. Note that gene-specific primers or alternate primers (T7 or T3 promoter primers) are used to achieve a minimum size of a 114-bp product. Step 2. Ligation of the PCR products. Step 3. Amplification of the ligated product to generate cDNA containing the desired mutation. Step 4. Restriction digestion of the final PCR product and ligation into the expression vector.