by Harald Zähringer, Labtimes 06/2012
Introducing mutations into genes is a crucial tool for protein structure and function analysis. A modified protocol of the quick-change method may facilitate mutation of multiple sites.
Many labs rely on the quick-change mutagenesis technique to introduce single mutations into double stranded (ds) plasmid DNA. The protocol calls for two complementary oligonucleotide primers harbouring the desired mutation, which are extended with Pfu polymerase leading to two nicked DNA strands. After digestion of the methylated, non-mutated parental strands, the nicked DNA strands are transferred into E. coli cells, which repair the nicks.
The quick-change protocol has also been adapted to multiple-site-directed mutations, however, with a few shortcomings: the multiple-site protocol is based on a different set of reagents to the single-site protocol, the efficiency is lower than 50% and last but not least, it is time consuming.
Hence, a group from Life Technologies thought of a better approach for multiple-site-directed mutations using the quick change technique and came up with an enhanced protocol (Liang et al., Anal. Biochem., 2012, 427, 99-101).
The group simply combines the PCR amplification of the original method with a recombination step to accomplish a fast and efficient multi-site-directed mutagenesis. The first step of the protocol is basically the same as for single-site mutations. Instead of one oligonucleotide pair, however, multiple oligonucleotide pairs are used as primers. To prepare for the recombination step, the mutation site in each oligonucleotide is flanked with at least ten unchanged nucleotides on both sides.
Let’s assume that you intend to mutate three sites. Though it is possible to put everything in one reaction tube and do a multiplex PCR, the authors recommend a three-tube strategy with three separate PCRs using the appropriate forward and reverse primers.
The primer sets in the individual PCRs are as follows: forward primer of site 3 and reverse primer of site 1; forward primer of site 1 and reverse primer of site 2; forward primer of site 2 and reverse primer of site 3 (please refer to the original paper for detailed instructions).
What you get after the PCR are three linear amplification products sharing homologous ends. The fragments are subsequently linked together by homologous recombination applying any commercially available recombineering kit based on dsDNA-repairing enzymes.
According to the authors, mutagenesis rates are independent of plasmid size and range between 90% and 100% for mutations that are more than 200 nucleotides apart from each other.
Last Changed: 10.11.2012