Description
The QuikChange site-directed mutagenesis method uses either miniprep plasmid DNA or cesium-chloride-purified DNA. The basic procedure starts with a supercoiled, dsDNA vector with an insert of interest and two synthetic oligonucleotide primers containing the desired mutation. The oligonucleotide primers, each complementary to opposite strands of the vector, are extended during temperature cycling by PfuTurbo DNA polymerase. On incorporation of the oligonucleotide primers, a mutated plasmid containing staggered nicks is generated. After temperature cycling, the product is treated with Dpn I. The Dpn I is used to digest the parental DNA template and select for the synthesized DNA containing mutations. Since DNA isolated from most E. coli strains is dam methylated, it is susceptible to Dpn I digestion, which is specific for methylated and hemimethylated DNA. The nicked vector DNA incorporating the desired mutations is then transformed into E. coli. The small amount of starting DNA template required to perform QuikChange mutagenesis, the high fidelity of the PfuTurbo DNA polymerase, and the low cycle number all contribute to the high mutation efficiency and decreased potential for random mutations.