The Stratagene QuikChange mutagenesis kit is an efficient, neat and rapid method for the mutagenesis of DNA. The product utilises pfu Turbo as a polymerase to replicate template DNA from complementary primers containing mutagenic nucleotides. This product may seem like straight forward PCR, however, amplification is 2n as opposed to 2
n (where n equals the number of cycles) because daughter DNA cannot act as template for subsequent rounds of PCR.
Using nanogram quantities of template, as few as 16 cycles of PCR can generate sufficient mutated product to allow simple inspection for product on an agarose gel. Observation of an addition band when compared to control reactions (including template) generally suggests a successful mutagenic event. The size of primer increases as the number of mismatched nucleotides within the primer increases (the more mismatches present, the larger the primer has to be) and the concentration of primers is always kept high to promote primer binding to template (and also to compensate for the unavoidable formation of primer dimers). Perhaps the most elegant part of the kit is the simple and efficient method of removal of the parental template. This step is achieved by digestion of reaction with DpnI. This restriction enzyme is a 'frequent cutter' that only digests methylated, hence template, DNA. Further, DpnI works effectively in the reaction buffer so there is no requirement to purify DNA at this step.
I have used this kit for simple site-directed mutagenesis with perfect success rates. For one, two and three nucleotide changes, primers of ~25 base pairs gave good extension. I have also used the technique to generate great libraries. Two codons, seperated by one unmutated codon, were completely randomised in primers of ~50 base pairs. Although mismatched base pairs must have been present in the DNA used to transform bacteria, this was of no concern, because although bacterial repair systems would correct the mismatch, a mutant is still a mutant!
As a cautionary tale, I did initially try to replicate the kit by buying the individual Strategene componenents and following the suggested protocols. Although this tactic worked in many cases, it was not 100% successful dispite varying many parameters. A Quik call to an extremely helpful and knowledgable Stratagene technical assistant revealed that batches of pfu Turbo can be rated for mutagenesis or PCR, the more efficient making the mutagenesis grade. This probably explained my mixed success and after ordering the complete kit, all my mutagenesis worked.
Refreshingly, this is another molecular biology product that lives up to its product specs. In one day, DNA can be mutated and transformed into bacteria. After another day, colonies can be sequenced and mutagenesis confirmed, further, only a handful of colonies need to be screened as removal of template is so efficient. The kit costs ~$400 for 25 reactions, and if you need to make more than that number of directed mutants then maybe a library is needed, which this kit can also generate.
Peter Haggie, Ph.D.
Postdoctoral Fellow
University of California, San Francisco