Site-directed mutagenesis using PCR-mediated introduction of silent mutations.

The alteration of a specific sequence of DNA using site-directed mutagenesis (SDM) provides an effective tool to probe the structure and function of gene products. With the advent of polymerase chain reaction (PCR), several new techniques were developed that facilitate SDM. An oligonucleotide containing mismatches at the desired site of mutation is used to generate a population of mutant fragments in the successive cycling. However, during the design of an appropriate methodology, several difficulties arise. The use of thermostable Taq DNA polymerase, which lacks proofreading ability, leads to a reduction in the fidelity of the amplified DNA (3). In addition, Taq DNA polymerase is known to place a 3′ overhang of a single base pair on amplified DNA, which often presents difficulties in cloning attempts. Most significantly, PCR is limited by the fact that sequence alterations must be located at the ends of the target sequence. To address this final concern, some methods of PCR-based mutagenesis have focused on the generation of two overlapping fragments containing the desired mutation. The fragments are generated with two complementary mutant oligonucleotides and one other oligonucleotide flanking each side of the target sequence. The methods are differentiated by the manner in which they seek to fuse these overlapping fragments. The fused fragment is successively subcloned using convenient restriction sites flanking the region of interest. One protocol, termed the “megaprimer” method, calls for PCRbased generation of a long mutant fragment that is subsequently used as a primer in a second round of PCR, extending the mutant fragment to the other side of the mutation (3,4,8). Such a method has been found to be technically difficult (5). More troublesome, however, is the fact that the singlestranded DNA overhang on the product of the first PCR often becomes misincorporated into the final product of the second PCR (9). Even using proofreading polymerases, which decrease the risk of undesired mutations in a single round of PCR, a great threat to fidelity is posed by the megaprimer method because it requires two full rounds of PCR, twice the number of cycles required by the method presented here. It has been suggested that if a convenient restriction site exists in the native sequence in the vicinity of the desired mutation, fusion of overlapping fragments can be carried out by ligating the ends generated by digestion (6). Similar methods calling for digestion using class 2S restriction enzymes have been developed (10). A recognition sequence incorporated into the end of the mutagenic primers allows for digestion downstream of the restriction site and fusion of the mutant fragments. However, significant drawbacks of such methods include the synthesis of costly, long primers and no allowance for screening of mutant plasmids because of the nature of class 2S enzymes, whereby the recognition site is lost after digestion. Often, silent mutations resulting from transformation of mutagenized plasmids have been used to aid in the screening of colonies by adding or removing restriction sites while leaving the protein product unchanged by taking advantage of codon redundancy. In an effort to expand the use of the silent mutation and avoid the difficulties of other SDM methods, we present a method for SDM that uniquely makes use of a silent mutation adding a restriction site. The method maximizes generation of mutated product and maintains its fidelity in several ways such as: (i) it requires only a single round of PCR, (ii) it allows for rapid screening at an early stage to confirm the presence of the mutation, (iii) it results in high-efficiency, sticky-end ligation and (iv) it minimizes the size of the PCR-derived fragment to avoid undesired mutations. It serves as a viable alternative to the class 2S method of SDM because it allows for cost saving on primer synthesis and screening of mutant products using the incorporated restriction site. The efficacy of this method is demon-