Stratagene’s QuikChange™ Site-Directed Mutagenesis Kit can be used for in vitro
site-directed mutagenesis to make point mutations, to switch codons, and delete or insert single or multiple bases or codons. These transformations can offer invaluable insight into protein structure and function.
The QuikChange™ Site-Directed Mutagenesis Kit is very quick (as the name says) and the protocol is easy to follow; it does not require specialized vectors, unique restriction sites, or multiple transformations. The kit contains enough reagents for 25 test reactions and 5 control reactions. The efficiency of the procedure is over 80% and the results are highly reproducible.
The kit is based on mutagenic primers that introduce specific mutations. The mutagenic oligonucleotide primers are designed by the researchers according to the mutation(s) they want to obtain; Stratagene provides recommendations regarding the optimal length, the melting temperature (Tm), optimal position of mutation(s) in the primer, and optimal GC content. For high mutation efficiency, primers need to be purified by FPLC or by PAGE. When setting up your experiments, it is advisable to thaw the dNTP mix (included with the kit) once and aliquot it to avoid repeated freeze-thaw cycles and to mix the components very well at all steps during reaction set-up.
The procedure utilizes a supercoiled double-stranded DNA (dsDNA) vector with an insert of interest and two synthetic oligonucleotide primers containing the desired mutation. The primers, which are complementary to opposite strands of the vector, are extended during temperature cycling by using PfuTurbo DNA polymerase (included with the kit). In the first step, the plasmid is denatured. Following denaturation, the oligonucleotide primers containing the desired mutation(s) anneal to the vector and are then incorporated to the synthesized strand of DNA, resulting in 2 nicked circular DNA strands. The temperature cycling used for synthesis and amplification is in accordance with the type of mutation desired (for point mutations, it is recommended to use 12 cycles; for single amino acid changes, 16 cycles; and for multiple amino acid deletions or insertions, 18 cycles).
Next, the methylated, non-mutated parental DNA template is digested with DpnI, an enzyme specific for methylated and hemimethylated DNA. (Note: DNA isolated from almost all E. coli strains is dam-methylated and therefore the parental vector DNA is susceptible to DpnI digestion, while DpnI does not digest the newly synthesized mutant DNA.) The circular nicked vector DNA containing the desired mutations is then transformed into Epicurian Coli® XL1-Blue supercompetent cells (included with the kit). Once transformed into the E.Coli, the nicks in the mutated plasmid are repaired during replication of the plasmid. Then the entire transformation reactions are plated on agar plates containing the appropriate antibiotic for the plasmid vector plus X-gal and IPTG for blue colony selection of positive mutants. The expected colony number is 50 to 800 colonies per plate. Low transformation efficiency could be observed if too much mineral oil is pipetted with the DpnI -treated DNA while transferring to the transformation reaction. Although it requires additional time and money, once you have identified and selected your colonies, it is always advisable to sequence the insert and to check the primer sequences for degradation/alteration.
I have used this kit to perform single point mutations in various proteins of interest. The kit is extensively used in my lab with a great rate of successful outcomes (most mutations are obtained with the first use of the kit). One example is the mutation I introduced into the Drosophila anillin gene by changing lysines 997- 999 to alanines in order to obtain transgenic flies whose NLS sequence was altered in anillin.
I highly recommend the Stratagene QuikChange™ Site-Directed Mutagenesis Kit for its high rate of success and ease of use.