These two novel polymerase chain reaction (PCR)-based methods enable efficient generation of substitution mutations in large plasmids (>10 kb). Site-directed mutagenesis is a common tool in the molecular biology field, allowing for a better understanding of the influence of DNA sequence on downstream biological and biochemical functions. The methods for performing site-directed mutagenesis vary but often include polymerase chain reaction (PCR) to generate point substitutions, deletions, or insertions in the DNA. The available PCR-based methods for generating point substitution mutations are typically the gold standard strategy. However, when using large plasmids, these methods present several disadvantages.
Overlap extension PCR methods involve the generation of overlapping DNA products with the desired mutations by two PCRs, subsequently used as a template for a final overlap extension. However, the final products are difficult to generate and dependent on cloning into a plasmid vector for expression, making this approach time-consuming and inefficient. An alternative and more straightforward approach incorporates complementary primer pairs with the substitution mutation at the center of each primer. Unfortunately, the formation of primer dimers is common, leading to reaction failure. Inverse PCR is another option, with one of the primers harboring the mutation near the 5’ end and the final product being phosphorylated and ligated after amplification. However, this approach is typically unreliable for large DNA templates, and DNA artifacts are frequent at the site of self-ligation. Advances are necessary for site-direct mutagenesis of large plasmids.
Researchers at the University of Florida have developed two novel PCR-based methods, “Sequential Single Primer Extension Reaction” (SSPER) and “reduce recycle PCR” (rrPCR), readily generating the desired site-directed mutations on large plasmids. These two methods are highly accurate, cost-effective, straightforward in primer design, quick, and applicable for both large and small plasmids.
PCR-based strategies for efficient, simple site-directed mutagenesis of large plasmids
These two PCR-based strategies, “Sequential Single Primer Extension Reaction” (SSPER) and “reduce recycle PCR” (rrPCR) readily generate desired site-directed modifications in large plasmids (> 10 kb). The methods incorporate easy removal of the primer(s) after the first reaction, allowing for the addition of a subsequent second reaction to generate and isolate the DNA product harboring the desired site-directed mutation(s). SSPER is a sequential design involving a single primer (p1) extension reaction followed by the removal of p1 and a similar single primer extension reaction with a second primer (p2) and the mixture of the original DNA template and newly synthesized ssDNA as the desired target.
rrPCR involves a pair of forward and reverse primers in a traditional PCR (PCR-1) style, designed to generate a PCR product of 0.1 to 1 kb with one or both primers harboring the substitution mutation in the center. Primers are not complementary, and the formation of primer dimers is minimal. After the first PCR, a simple PCR cleanup removes the primers. The resulting mixture is reduced in primers and recycled for a second PCR (PCR-2), with the dsDNA product of PCR -1 serving as the primer pair and the recycled plasmid DNA as a template again. In both cases, methylated DNA plasmids serve as templates, being removed by DpnI digestion at the end of the reactions prior to transformation into E. coli.
