Site-Directed Mutagenesis of Large (13-kb) Plasmids in a Single-PCR Procedure

Although there are many methods for site-specific mutagenesis, most are only applicable for short (< 7-kb) plasmids. The few methods that have been developed for longer plasmids are complicated procedures that involve multiple PCR steps or complex recombination and ligation reactions (2,4–6). Here, we provide a simple single-PCR procedure for site-specific mutagenesis of long plasmids (13 kb), even with primers having strong secondary structure. Our protocol is a modification of the QuickChangeTM Site-Directed Mutagenesis (SQM) kit (Stratagene, La Jolla, CA, USA) method originally published by Weiner and Costa (7). The pair of complementary oligonucleotides containing the desired mutation must (i) be 30–45 bases long, (ii) contain at least one C or G base at each terminus, (iii) have melting temperatures of at least 78°C and (iv) have the mutations in the middle of the primers so that there is at least 10 bases of sequence complementary to the parental sequence on either side. The oligonucleotides must also be purified by either SDS-PAGE or HPLC. Using unpurified oligonucleotides drastically reduces mutagenesis efficiency (data not shown). In addition, for mutagenesis of large plasmids, we found it is optimal to design primers that have little or no secondary structure. This is an important criterion that is not accounted for in the SQM kit. The PCR mixture contains 150 ng each oligonucleotide, 10 ng template plasmid DNA, 200 μM dNTPs, 2.5 U PfuTurboTM DNA polymerase (Stratagene) and the buffer supplied with the polymerase in a total volume of 50 μL. PCR is performed under the following conditions: denaturation at 95°C for 30 s, followed by 16 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 1 min and extension at 68°C for 2 min/kb template (which is identical to that described in the SQM kit). A portion of the PCR mixture is then examined by agarose gel electrophoresis to determine whether the correct-sized product was obtained. If these PCR conditions did not generate the correctsized product, then several parameters of the PCR can be altered to produce the correct product. When the correct-sized product is obtained, then the PCR product (50 μL) is incubated with 20 U DpnI for 2 h at 37°C. This latter step is critical because it preferentially digests the parental DNA (which is methylated and therefore is subject to DpnI digestion) and thereby increases dramatically the frequency of clones with the desired mutation (3). Five microliters of the DNA are then transformed into 100 μL competent Top10® E. coli cells (Invitrogen, Carlsbad, CA, USA). To test the method, we used a 6.2-kb plasmid (β-299) and a 13-kb plasmid (β-290), which contain a 3.2-kb SalI/ BamHI mouse T-cell receptor genomic fragment inserted into the 3-kb Bluescript® (KS+) vector (Stratagene) and pHβAPr1-neo vector (EV-107) of approximately 9.8 kb, respectively (1). The primers used for mutagenesis are shown in Table 1. There are three major differences between our protocol and that of the SQM kit that we found were critical for generating large PCR products (13 kb) mutated at high efficiency. (i) The inclusion of 5%–10% dimethyl sulfoxide (DMSO) in the PCR mixture was often necessary when using primers with strong secondary structure and/or when the plasmid template was greater than 6 kb. (ii) The temperature of the annealing step of the PCR sometimes needed to be altered depending on the secondary structure of the oligonucleotides used for amplification. We found that annealing temperatures between 55°C and 72°C generated visible products for most oligonucleotides. (iii) We used excess DpnII (20 U) for a longer incubation (2 h) than suggested by the SQM kit to completely digest the parental DNA. We found that this step was critical for providing a higher efficiency of mutagenesis (95%–100%) of large plasmids (13 kb) compared to the more modest mutagenesis efficiency (about 80%) achieved by the standard SQM kit for smalland intermediate-sized plasmids. After following the procedure described above, we sequenced plasmid DNAs prepared from individual bacterial colonies. We found that the 6.2-kb plasmid template efficiently generated PCR product, and least 92% of the DNA clones contained the desired mutation (Table 2). In contrast, PCR conditions required to amplify the 13-kb plasmid typically had to be modified to generate the correct-sized PCR product. For the oligonucleotide pair P1, each primer of which has weak secondary structure, we found that it was only necessary to include 10% DMSO to generate the correct-sized PCR product. [Secondary structure can be determined by using the formula obtained from the Genosys Web site (www. genosys.com)]. Ninety-five percent of the clones obtained from this PCR product were correctly mutagenized (Table 2). When we used oligonucleotide pairs P2 and P3, which have moderate and strong secondary structure, respectively, we could not generate detectable PCR products even with DMSO. To obtain the correct-sized PCR product with these oligonucleotides, we raised the annealing temperature to 72°C (8°C–10°C lower than the Tm of the primers) and increased the denaturation time to 45 s per cycle. These P2 and P3 PCR products also generated a high percentage of correctly mutagenized clones (Table 2). However, we found that less PCR product was generated using moderate-tostrong secondary structure primers, as compared with using primers with weak secondary structure (data not shown). Consistent with the generation of less PCR product, we found that transformation of this DNA yielded 7–15 times fewer colonies. Our method does not require using the SQM kit. Although the kit is useful for beginners, we found that the kit did not work with large (> 10 kb) plasmids, according to the manufacturer’s protocol (data not shown). Furthermore, our method is cheaper and requires less specialized reagents. For example, the kit includes Epicurian Coli® XL1-Blue Supercompetent bacterial cells (Stratagene), which are expensive, sensitive to freezing and thawing, and require particular conditions for transformation [e.g., the use of NZY+ broth (Stratagene) and Falcon 2059 tubes (Becton Benchmarks