Generation of deletion and point mutations with one primer in a single cloning step.

Site-directed mutagenesis is an important technique for studying DNA and protein function. With the advent of PCR, several new approaches have been successfully developed (1,3,5–9). Early methods involved two-step PCR followed by subcloning, which is time consuming and error prone. Recently introduced site-directed mutagenesis kits such as QuikChangeTM Site-Directed Mutagenesis Kit (QCS) developed by Stratagene (La Jolla, CA, USA) is a widely used PCR-based system (4) that eliminates the necessity to subclone the amplified DNA fragment. It requires two complementary mutagenic oligonucleotide primers and uses a plasmid to amplify the desired product by highfidelity Pfu DNA polymerase (Stratagene). Following temperature cycling, the product is treated by the restriction endonuclease DpnI (Stratagene) that will cut only fully or hemimethylated 5′-Gm6ATG-3′ DNA sequences. Thus, only the original parental methylated plasmid DNA will be digested. The remaining unmethylated and nicked double-stranded plasmid incorporating the desired mutations is then transformed into Epicurian Coli® XL-1Blue supercompetent cells (Stratagene). The yield of clones harboring mutations is very high and varies, depending on the template length, from 80% to more than 90% for 2.9-kb plasmids (4). Methods for generating internal deletions have been described by using inverse PCR, but these involved two steps (9). A simplified method for deletion mutagenesis is used in the ExSiteTM mutagenesis kit by Stratagene, but this method also involves the use of phosphorylated primers and a ligation step. The use of the QuikChange mutagenesis kit has been reported for introducing deletions or insertions of only 12 bp (4). Here, we present a significantly improved method based on the QuikChange system for generating deletion and point mutants. This method allows the use of a single primer to generate deletions exceeding 3000 bp and point mutations in a single linear amplification reaction. In our laboratory, we routinely use the QuikChange kit for point mutations. However, it was not clear how this protocol could be applied for deletions of more than 12 bp (4). We used a plasmid containing Pals1 cDNA (total length 6.7 kb) as a template to make several deletion and point mutants (2). The PCR mutagenesis reaction was performed in a reaction volume of 25 μL that included 1 μL plasmid DNA (in TE or water) from Wizard® Plus SV Minipreps DNA Purification System by Promega (Madison, WI, USA) or from QIAprep® Spin miniprep kit by Qiagen (Valencia, CA, USA) miniprep (100– 300 ng/μL) template DNA, 200 nM each primer, 200 μM dNTPs and 1.25 U Pfu enzyme in 1× Pfu DNA polymerase reaction buffer. A preliminary step of denaturation at 95°C for 3 min was followed by 18 cycles of PCR. These PCR cycles consisted of 15 s of denaturation at 95°C, 1 min of annealing at a temperature 2°C higher than the melting temperature (Tm) of the head of the primer to increase specificity of amplification (see below) and 12 min of extension at 68°C using a PTC200TM thermal cycler (MJ Research, Watertown, MA, USA). In cases when the Tm exceeded the extension temperature, we applied a two-step PCR annealing and extending of 68°C. Finally, the PCR product was treated with 1 μL DpnI endonuclease for 2 h at 37°C and 2 μL DpnI-digested DNA was transformed into 50 μL Epicurean Coli XL1 Blue supercompetent cells. The number of kanamycin-resistant clones varied from 100 to 2000. We experimented with primer pairs that would result in the highest yield of the desired deletion. We routinely analyzed clones by PCR using primers flanking the deletions and obtained efficiencies of between 25% and 70%. Selected clones were sequenced and confirmed the PCR results. Table 1 summarizes different examples of primer pairs or single primers used in the mutagenesis experiments. For simplicity, the 5′ part of the primers complementary to the region upstream of the introduced deletion we called the tail, and the 3′ portion of the primer complementary to the region downstream of the deletion we called the head. As shown in Table 1, when using primer pairs of different design, deletions of up to 3 kb could be introduced into the plasmid by this simple one-step protocol. We believe that the same approach could be applied to any sequence because we successfully deleted several regions of the coding Benchmarks