Site directed mutagenesis by ligation‐during‐amplification: Change‐IT Multiple Site Directed Mutagenesis Kit

Current site directed mutagenesis methods suffer from low mutagenesis efficiency when faced with large templates, large deletions, or multiple mutations. We sought to address these deficiencies by amplifying plasmid template and ligating the product into closed circles at the end of each round of PCR. This was accomplished by including in the reaction mixture a NAD + ‐dependent thermostable ligase and FideliTaq™ DNA Polymerase, USB Corporations’ proofreading enzyme blend, and by designing the primers to minimize primer dimer formation. These measures allowed us to create single, double, and triple mutations with 90, 80, and 70% efficiency, respectively. Deletions of 300bp were also able to be created with somewhat lower efficiency. Finally, mutations in plasmid templates greater than 8kb were able to be created on a routine basis. Very large plasmids though required additional ligase and FideliTaq™ in the reaction mixture during amplification. In summary, we have developed a method for the efficient creation of oligonucleotide‐directed mutations in plasmids that allows multiple mutations, large deletions, and large templates to be created with ease.