Site-Directed Mutagenesis Mediated by a Single Polymerase Chain Reaction Product

Site-directed mutagenesis (SDM) by deletion of a unique restriction site, introduced by Deng and Nickoloff (1 ), allows site-specific mutagenesis of a plasmid DNA without any subcloning steps. This procedure uses two mutagenic primers: one carries the desired mutation; the second, acting as a selection primer, carries a mutation in a unique, nonessential restriction site in the target plasmid. The method relies on the simultaneous annealing of two primers (mutagenic and selection primers) to one strand of the denatured double-stranded plasmid DNA. After DNA elongation and ligation, plasmids lacking the selection-restriction site are expected to encode the desired second site mutation. This strategy is efficient on a substantial number of templates; however, the authors have found that, in the case of some plasmids, the recovery of the desired mutation is much lower, yielding the desired mutant products at a frequency of less than 10%. The reason for this low efficiency may stem from the nucleotide sequence of the target DNA, which may acquire stable secondary structures, such as stem-loops. These nucleotide structures may interfere with the annealing of the mutagenic primers, and could result in low yields of the mutant plasmids.