Holly Hogrefe, Ph.D.
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.
Continued Innovation Leads to Lightning Fast Enhancements
While the site-directed approach described above represent a marked improvement over PCR-based methods, within the last five years, new methods were introduced to address the demand for faster workflows and increased productivity. What distinguishes some of the newer methods from their predecessors was the introduction of a new enzyme, Pfu fusion DNA polymerase. Pfu fusion’s secret: tighter binding and increased processivity through the presence of a C-terminal DNA binding domain, in order to enable faster PCR cycling. In addition, optimized protocols and reagents reduced the length of the PCR and DpnI selection steps, providing up to a three-fold reduction in overall mutagenesis turn-around time.
In addition to the improved site-directed mutagenesis method described above, a newer approach was developed with the ability to create mutations at multiple sites simultaneously. While error-prone PCR-based methods are typically limited to two simultaneous mutational sites, by employing a unique multi-enzyme polymerase blend, it is possible to introduce mutations at up to five sites simultaneously. Conventional techniques to perform site-directed mutagenesis at five sites (along with accompanying miniprep and sequencing reactions) can take up to 15 days to complete. With multi-mutational methods, it is possible to reduce this time to about three hours.
Synthetic Biology as the Next Step in Site-Directed Mutagenesis
The efficiency and ease-of-use of site-directed mutagenesis methods have facilitated engineering of promoter and coding regions of numerous genes. Continuing improvements to Pfu polymerase have led to enhancements, permitting protocols with higher fidelity, longer length-capability, multi-site targeting, and significantly faster time-to-completion. Looking forward, leading the drive for continued innovation in the area of mutagenesis means embracing novel technologies, which may yield new enhancements in speed, efficiency, fidelity, or throughput. One such area, synthetic biology, may enable broad scanning mutagenesis approaches and permit the sequential adding of mutations through a domain in order to understand the role of each amino acid in a protein, find improved variants, and outline structural linkages. Synthetic biology approaches allow the scaling up of mutagenesis experiments with accompanying significant reductions in time and cost for large-scale assays.
For more on mutagenesis, be sure to check out “From Conventional Mutagenesis to Complex, Rationally Designed Mutant Libraries: Enabling New Opportunities in Scalability“.
Holly Hogrefe, Ph.D., is NGS R&D Director at Agilent Technologies.