In vitro site-directed mutagenesis is an invaluable technique for characterizing the dynamic, complex relationships between protein structure and function, for studying gene expression elements, and for carrying out vector modification. Several approaches to this technique have been published, but these methods generally require single-stranded DNA (ssDNA) as the template and are labor intensive or technically difficult. Our QuikChange Lightning Site-Directed Mutagenesis Kit allows site specific mutation in virtually any double-stranded plasmid, thus eliminating the need for subcloning and for ssDNA rescue.  In addition, the QuikChange Lightning Site-Directed Mutagenesis Kits do not require specialized vectors, unique restriction sites, multiple transformations or in vitro methylation treatment steps. The simple, rapid three-step generates mutants with high efficiency.

Using the most advanced high fidelity enzyme technology, the protocols have been accelerated while maintaining the highest accuracy for site-directed mutagenesis (see Figure 1). Exclusive to the QuikChange Lightning Site-Directed Mutagenesis Kit is a proprietary Pfu-based polymerase blend and the newly optimized Dpn I enzyme, which together allow for mutagenesis in approximately one hour, plus an overnight transformation.

The QuikChange II site-directed mutagenesis kit is used to make point mutations, replace amino acids, and delete or insert single or multiple adjacent amino acids. The QuikChange II site-directed mutagenesis method is performed using PfuUltra high-fidelity (HF) DNA polymerase for mutagenic primer-directed replication of both plasmid strands with the highest fidelity.   The basic procedure utilizes a supercoiled double-stranded DNA (dsDNA) vector with an insert of interest and two synthetic oligonucleotide primers, both containing the desired mutation (see Figure 1).  The oligonucleotide primers, each complementary to opposite strands of the vector, are extended during temperature cycling by PfuUltra HF DNA polymerase, without primer displacement. Extension of the oligonucleotide primers generates a mutated plasmid containing staggered nicks. Following temperature cycling, the product is treated with Dpn I. The Dpn I endonuclease (target sequence: 5´-Gm6ATC-3´) is specific for methylated and hemimethylated DNA and is used to digest the parental DNA template and to select for mutation-containing synthesized DNA.6 (DNA isolated from almost all E. coli strains is dam methylated and therefore susceptible to Dpn I digestion.) The nicked vector DNA containing the desired mutations is then transformed into competent cells.