Highly efficient and simple SSPER and rrPCR approaches for the accurate site-directed mutagenesis of large and small plasmids

Advances are needed in the site-directed mutagenesis of large plasmids for protein structure-function studies, as current methods are often inefficient, complicated and time-consuming. Here two new methods are reported that overcome these difficulties, namely the single primer extension reaction (SSPER) strategy that reaches 100% efficiency and the reduce recycle PCR (rrPCR) method that is advantageous in generating single and pairwise combinations of mutations. Both methods are distinguished from current technologies by the addition of a step that easily removes the oligonucleotide primer(s) after the first reaction, thus allowing for the addition of a second reaction in chronological sequence to generate and isolate the appropriate DNA product with the sitedirected mutation(s). High efficiency of the methods is demonstrated by generating single and paired combinations of the 11 site-directed mutations targeted on 5 different plasmid DNA templates ranging from 10 to 12 kb and 57-60% GC-content at a rate of 50-100%. Overall, the methods are demonstrated to be (i) highly accurate, allowing for screening of plasmids by DNA sequencing, (ii) streamlined to generate the mutations within a single day, (iii) cost-effective in requiring only two primers and two enzymes (DpnI and a proofreading DNA polymerase), (iv) straightforward in primer design, (v) applicable for both large and small plasmids, and (vi) easily implemented by entry level researchers.

Automated summary

This study reports two new methods for site-directed mutagenesis of plasmids, which overcome the current issues of low efficiency, complexity, and time consumption. Both methods have high efficiency and can generate and isolate the appropriate DNA products with site-directed mutations.