Site-Directed Mutagenesis: Conventional Mutagenesis with Limited Scale
Conventional site-directed mutagenesis methods let you rapidly and efficiently create point mutations, amino acid substitutions, insertions, and deletions in virtually any double-stranded plasmid—useful for understanding protein-protein interactions and structure-function relationships. These methods are also commonly used for functional validation of genetic variants such as SNPs, codon optimization to increase protein expression, and for knocking-out undesired activity. However, because of the targeted approach, prior knowledge of the protein’s structure is required to achieve the desired mutation. In addition, researchers who wish to increase the scale of mutagenesis (beyond single-site mutations) may find these methods limited.
Multi-mutagenic methods such as QuikChange Lightning Multi, addresses some of these limitations by permitting rapid and efficient creation of point mutations in plasmid DNA at up to five sites simultaneously. While PCR-based methods are typically limited to two simultaneous mutational sites (each targeted with one of the PCR primers), by employing a unique multi-enzyme polymerase blend, multi-mutagenic methods are capable of incorporating up to five mutagenic oligos simultaneously in about three hours versus up to two weeks when using PCR-based methods. Such methods can be useful for identifying combinations of causal mutations that behave synergistically. Moreover, with a one-primer-per-site protocol, these methods can be used for codon saturation experiments, which employ degenerate codon (NNK; where N=AGCT; K=G/T) primers to explore the impact of all possible side chain replacements. However, like its site-directed counterparts, multi-mutagenic approaches also require some prior knowledge of the protein’s structure and its multi-site feature can be limited by a lack of knowledge of which positions affect function. Finally, while the Multi method can be scaled up to cover a complete domain, it could require tens to hundreds of reactions.