by Adam Clore, Director of Development & Innovation, Integrated DNA Technologies
Site Directed Mutagenesis (SDM) is an invaluable tool that allows scientists to alter many characteristics of proteins. The method, however, introduces several challenges. In most cases, changing the function of a protein cannot be precisely determined a priori, often necessitating that a large number of mutants be generated and screened to find one containing the desired traits. Performing SDM across many sites is often complicated due to the secondary structure of DNA. Not all sites are created equal, particularly if they’re highly structured, have a lot of repeat sequences or have extremes in guanine-cytosine (GC) content.
To overcome these inherent challenges, it’s important to consider the appropriate scale of the experimental design to ensure enough variation is present to find the desired trait while minimizing the time and cost in creation and screening of mutant libraries. While there are some decent predictive models, particularly for well characterized proteins like antibodies, they are not 100% accurate, so finding the desired mutant often involves large libraries or iterative rounds of mutagenesis and screening depending on the size of the protein and the resources available in the lab.
The best solution lies in identifying the most efficient and effective methods. Improving a few key steps in the process can increase the chances of success in SDM.
Primer-based SDM
Mutagenesis is often done in plasmids, which are circular pieces of DNA. Small changes in one or two bases can be done easily by using two primers that have one or more changes built into them. The primers incorporate the changes into the final product during amplification and can be ligated, cloned, and screened after PCR. These changes can be discreet, changing one sequence to another sequence, or they can be variable, changing one sequence to a variety of sequences by incorporating several different primer sequences or a primer with mixed bases.
Primer design basics
The optimal design of primers in SDM experiments can make or break your experiment. The 3’ end of the primer, starting after the mutation site and extending to the end of the primer, is most critical in determining specificity and efficiency. Researchers should ensure this region has a Tm that is at least 55°C. The addition of a “GC clamp,” that is, ending the primer with two or more Gs or Cs, can also increase efficiency of extension.
Figure 1: Inverse PCR for a substitution. One of the two primers contain the mutation of interest (indicated by the blue bubble). In this case, both primers contain 5’ phosphorylated ends to allow the two ends to be ligated together following amplification. PCR is used to amplify the entire circular plasmid to create a linear template that contains the substituted sequence. This fragment is then circularized by intramolecular ligation and the resulting plasmid is transformed into host bacteria for propagation.
Ideally, primers will be designed in areas that lack a lot of repeats or secondary structures like hairpins. If it’s not possible to avoid those complexities, one way to get around them is to extend the length of the primers in the 3’ direction. There are analytical tools available that can look for hairpins and other complexities in sequences of DNA.
To incorporate more than one change at a time, it’s also possible to order mixes of oligos that are tailored to specific experiments. For example, researchers can order a tube that contains hundreds of oligos with changes in one area. The primer is the same except for point mutations within the different variants. Using a mix instead of a single primer for cloning results in several different changes that are localized in one area.
Avoiding pitfalls
Setting up the ideal PCR reaction parameters for SDM is key to avoiding mistakes that can set projects back. Here are the key elements to consider:
- Temperature: Optimizing the annealing temperature will prevent nonspecific primers from binding. Keeping the denaturation temperature between 94 and 95 degrees Celsius ensures complete denaturation and proper polymerase activity. Increasing annealing temperature can help if nonspecific amplification is observed and decreasing annealing temperature can help when little or no amplification happens.
- Time: Extension times that are too short will result in little or no product, and the initial denaturation time should be longer than subsequent steps to ensure proper activity and amplification. Different polymerases require different extension times. Check with your manufacturer’s instructions for the optimal extension times for your polymerase
- Cycle number: The optimal approach for SDM is to use the minimum number of cycles needed to produce a detectable band when one-tenth of the reaction is analyzed.
After primer design and PCR optimization, don’t forget the DpnI digestion. DpnI is an enzyme that digests methylated DNA grown in bacteria. After DNA is amplified from the plasmid and changes are incorporated into it, DpnI is used to digest away the original plasmid DNA. This is an important step because supercoiled plasmid DNA often transforms with higher efficiency and can overwhelm your experiment if not removed prior to transformation and screening.
Creating large changes to proteins
Large-scale mutagenesis—adding or replacing entire motifs to a gene, thereby changing the functional part of a protein—requires different methods of mutagenesis. These larger changes require copying or linearizing the portion of the plasmid that’s being preserved. That creates a plasmid backbone, which holds a portion of a sequence that can be added to or changed, often with a completely synthetic sequence.
The best way to do this is with double-stranded DNA fragments that can be ordered in varying lengths to match the motif that’s being replaced. That makes it simple to ligate in the new DNA or use any modern assembly technique to insert the new piece into the plasmid.
The value of automation
Incorporating automation into SDM can greatly increase the speed of experiments as well as their reproducibility. In the early days of synthetic biology, researchers spliced genes together with restriction enzymes, but changing the strings of As, Ts, Gs, and Cs was complex and time consuming. Automation has made it much easier to introduce novel sequences into cells.
Automated colony pickers, liquid handlers, robotic arms, and other tools allow scientists to spend less time picking bacterial colonies and pipetting liquids so they can devote more time to finding creative solutions to problems and perfecting SDM experiments.
This equipment, which used to be cost-prohibitive for academic labs and even some startups, is now more accessible than ever. Automation can greatly increase the number of sequences researchers can mutagenize as well as the number of colonies that they’re screening.
Reaching the full potential of SDM
There are several different ways to create variations and introduce mutagenesis, and researchers shouldn’t get locked into any single method. While $10 worth of primers may be enough to perform a small SDM experiment, spending $89 on a double-stranded DNA fragment, and replacing a whole section, can ultimately save money in sequencing and other reagent costs. Avoiding one-size-fits-all approaches is the key to realizing the full potential of SDM. For more tips and strategies, download the IDT Mutagenesis Handbook, which includes an overview of experiment applications, protocols, and troubleshooting.
About the Author: Adam Clore is Director of Development & Innovation at Integrated DNA Technologies. For 35 years, IDT’s innovative tools and solutions for genomics applications have been driving advances that inspire scientists to dream big and achieve their next breakthroughs. IDT develops, manufactures, and markets nucleic acid products that support the life sciences industry in the areas of academic and commercial research, agriculture, medical diagnostics, and pharmaceutical development. For more information, visit www.idtdna.com.