QuikChange® Multi Site-Directed Mutagenesis Kit From Stratagene

Think about this: You want to generate three mutations in the same gene. With the traditional QuikChange® Mutagenesis Kit, it would involve three sequential mutagenesis reactions. That means the amplification, digestion and transformation, plus sequencing. Imagine that you can do it all in a single reaction with an efficiency that approaches that of the single mutagenesis kit.

Stratagene’s QuikChange® Multi-Site Directed Mutagenesis Kit can do that and much more, and very efficiently. How does it work? The system is an adaptation of the original QuikChange® method with a few modifications: The novel technology of the QuikChange® Multi system allows mutagenesis at multiple sites in a single round, using a single oligonucleotide per site.

First, a thermal cycling procedure is used to synthesize a single mutant strand. (All oligonucleotides are designed to bind the same strand of the template DNA.) PfuTurbo DNA polymerase then extends the mutagenic primers with high fidelity and without primer displacement, generating ds-DNA molecules with one strand bearing multiple mutations and containing nicks. The nicks are sealed by components in the enzyme blend.

In the second stage, the thermal cycling reaction products are then treated with the restriction endonuclease Dpn I. The DpnI endonuclease is specific for methylated and hemimethylated DNA and is used to digest the parental DNA template. DNA isolated from almost all E. coli strains is dam methylated and therefore, susceptible to digestion.

Finally, the reaction mixture, enriched for multiply mutated single stranded DNA, is transformed into XL10-Gold ultracompetent cells, where the mutant closed circle ss-DNA is converted into duplex form in vivo. Double stranded plasmid DNA may then be prepared from the transformants and analyzed by appropriate methods to identify clones bearing each of the desired mutations.

The whole reaction, including the transformation, can be performed in a single day so in theory, multiple mutations in the same gene can be obtained in a matter of a few days (considering the time needed to grow the bugs, prep the DNA and sequence different clones). The system can also be used to randomize key amino acids using oligos containing degenerate codons. A collection of mutants can be created in a single QuikChange® Multi kit reaction and then assayed for mutant activity using any appropriate functional screen.

We used the QuikChange® Multi-Site Directed Mutagenesis Kit to generate up to three simultaneous mutations in different vectors and inserts. Most of the time, the efficiency was close to 100% for the three mutations in the same clone. Stratagene claims that it is possible (and it is theoretically possible), to obtain combinations of 1, 2 or 3 mutations when you are trying to generate a triple mutant, but we have never observed that. (It should be possible to adjust the mutation efficiency at each site by varying primer concentrations.) The kit provides a control template and primers that can be monitored using blue/white screening but we believe that it is not necessary to perform the control reaction since in most of the cases, the reaction works very efficiently. Primer design instructions are very straightforward, but should be followed carefully. Primers may be designed to bind to adjacent sequences (as long as they don’t overlap) or to well-separated regions on the same strand of the template plasmid . In general, we find that using less template than recommended (25 ng instead of 50 ng) increases the mutagenesis efficiency.

In summary, this is a highly recommended product that allows you to generate up to 5 mutations simultaneously in a simple protocol that takes only one day. The success rate is extremely high and usually screening 4 colonies is enough to find the desired mutation.