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What is site-directed mutagenesis?
Mutagenesis can largely be placed into two categories—site-directed or random. Random mutagenesis is used to generate a variety of untargeted mutations or to quickly screen for mutations across a genome. Site-direct mutagenesis is used to generate mutations in a specific or targeted way to a known “site” or sequence within a genome.
As discussed in the DECODED Site-directed mutagenesis methods, site-directed mutagenesis can be completed using a variety of approaches depending on the goal of your experiment. Traditional site-directed mutagenesis by PCR is completed using oligonucleotides designed to generate changes (insertions, deletions, or terminal additions) to small sequences of DNA.
Here, we look at some common issues found during PCR-based site-directed mutagenesis and discuss a few tips for troubleshooting your site-directed mutagenesis experiment.
Troubleshooting your PCR-based site-directed mutagenesis experiment
- Check your primer design
As mentioned above, oligonucleotide primers are the starting point for PCR-based site-directed mutagenesis. Carefully designed primers will help ensure that you achieve the intended mutation in your target sequence. Poorly designed primers can result in no amplification, truncated amplification products, or multiple amplification products after your initial PCR.
For help with site-directed mutagenesis primer design, try IDT’s OligoAnalyzer™ Tool. This tool allows researchers to design new primers and evaluate the physical properties of your existing primers. Check out the DECODED Determining the physical characteristics of your oligos to learn more about the utility of the OligoAnalyzer Tool.
- Check template quality and concentration
Another common issue in any PCR is the quality and/or concentration of the template DNA (what you’re amplifying/mutating) being used. If too much template is used, the PCR might not produce as much product as you need, or multiple products might be generated. Similarly, if the concentration of template is too low then the resulting PCR product may not be enough for downstream reactions.
The quality of your PCR reaction can always be checked using gel electrophoresis; a high-quality PCR will result in crisp, clear band(s) on your gel. A PCR with imbalanced/low-quality template DNA will have either faint band(s), indicating little product, or smeared/multiple bands, which indicates the need to decrease the concentration of template DNA.
- PCR conditions
It can be difficult to pinpoint exactly where an experiment went wrong and what caused it to fail. One easy way to narrow down the list of potential issues is to include both a positive control and no-template negative control in all PCRs. This, in addition to using a commercially available master mix or mixing your own master mix (commonly used in all PCRs), can minimize variations between reactions as well as determine if the reaction failed due to a reagent issue (e.g., a reagent is expired or was accidently left out).
Reagent concentrations and PCR cycling conditions are also important to troubleshoot. Similarly, to the template concentrations discussed above, imbalanced concentrations of reagents within your PCR will result in a less efficient PCR and low-quality product. PCR cycling conditions such as cycle number, annealing temperature, extension time, etc. all require optimization and can change depending on the primers, template DNA, and master mix being used.
- Ligation reaction
Once you’ve successfully obtained an optimized PCR product and digested your plasmid, the next step for PCR‑based site-directed mutagenesis is the ligation reaction. Here, the ratio of high-quality PCR product (DNA insert) to plasmid is very important. The key is to use a concentration of insert that is high enough to yield enough product for an efficient transformation reaction but low enough to avoid generating large intermolecular ligation products.
The resulting product of a ligation reaction can be confirmed through gel electrophoresis, keeping in mind that these reactions rarely finish so some linear DNA is expected to appear on the gel.
- DpnI digestion
Whether you need to complete a DpnI digestion reaction depends on a number of factors. Importantly, DpnI targets methylated DNA therefore if the plasmid or DNA being used in your site-directed mutagenesis reaction is not methylated then you do not need to complete a DpnI reaction.
If you are working with methylated DNA then this enzyme can be used to digest the wild-type (untransformed) DNA left after your initial PCR and ligation steps. You can check the efficacy of your DpnI digestion by transforming your cells with DpnI-digested DNA and DNA not digested with DpnI. The number of colonies on your plate with the DpnI-digested DNA should be roughly half the number of colonies obtained with the DpnI-free DNA.
- Transformation efficiency
Transformation of competent cells should be done carefully as these cells are extremely fragile. They should be kept on ice throughout the transformation protocol and pipetting should be done slowly to avoid damaging cells. It is also recommended that you always double-check your cells’ heat-shock protocol, follow it carefully, and pay attention to the tubes in which you carry out this step.
Other recommendations for a successful cell transformation step include:
- Desalt DNA to control for salts in your transformation reaction.
- Carefully select antibiotics/concentrations to select against untransformed cells.
- Monitor cells for signs of contamination (e.g., odd culture color, unusual clumping).
- Select only appropriate competent cell strains, this can vary depending on the size of your insert, etc.
- Be aware of toxic sequences—i.e., sequences that when expressed in your competent cells result in a protein that is toxic to your cells.
Additional tips for site-directed mutagenesis by PCR
Finally, it is worth noting that most of the items listed above can be negatively impacted by reaction inhibitors which could cause your reaction to fail or at least reduce reaction efficiency. Additionally, it is recommended that you check all your enzymes and reagents to ensure you are storing them at optimal temperatures to prevent the loss of activity.
For more information about reaction inhibitors, troubleshooting your site-directed mutagenesis experiment, and review a site-directed mutagenesis protocol, download The Mutagenesis Handbook and review Section 5: Troubleshooting.
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