Mutagenesis experiments allow researchers to modulate protein activity and characterize structure- function relationships, which enriches our understanding of basic cellular processes and disease mechanisms and fuels discoveries in new therapies for complex diseases such as cancer. Site-directed mutagenesis is the method of choice for altering a gene or vector sequence at a selected location. Point mutations, insertions or deletions are introduced by incorporating primers containing the desired modification(s) with a DNA polymerase.
Today’s conventional site-directed mutagenesis methods, first introduced in 1995 (see Strategies 9(1): 3-4 under “Resources”), typically utilize a three-step, one-day method to introduce point mutations, amino acid substitutions, deletions, and small insertions in virtually any double-stranded plasmid template with high rates of efficiency. The success and prevalent adoption of these conventional methods can be attributed to the high rate of efficiency (>80% colonies with desired mutation) of such assays and the relative ease of use. Continued improvements in these assays in the following decade led to enhancements in fidelity, robustness, and the number of sites that can be mutagenized simultaneously.
Site-directed mutagenesis methods such as the QuikChange method have a number of advantages over PCR-based approaches. For example, the frequency of unintended errors is higher in PCR strategies (copies are copied), which increases the amount of sequencing and error correction required downstream. In contrast, site-directed methods may employ a linear amplification approach (only parental strands are copied) in addition to high-fidelity Pfu polymerases, to minimize the number of clones with undesired mutations. This is reflected downstream, by the need for less diagnostic sequencing of clones and correction of clones with incorrect mutations. Additionally, PCR-based strategies have been hampered by laborious cloning steps and low efficiencies (e.g., long range PCR of vector backbones); a contrast to the site-directed method’s >80% efficiency and three-step one-day protocol. Finally, robustness, or the ability to introduce mutations in larger templates, has been improved through the introduction of ultra-high-competency cells.