The use of restriction enzymes to characterize DNA has been popular since the 1970s. Today, this “old school” technique is still one of the easiest and fastest ways to assess DNA sequences.
Like most lab reagents, restriction enzymes can be fickle and you should bear a few things in mind when using them. Generally, sticky-ended enzymes have greater digestion efficiencies than blunt-ended enzymes. They also tend to be commonly used for cloning applications since they may also reduce the occurrence of vector self-ligation.
Whether you are simply digesting a plasmid to identify it, performing complicated cloning or screening applications, restriction digests require a considerable amount of thought! Let’s look at the main considerations:
1. Are You Using the Right Conditions?
Every enzyme is different and each requires specific conditions, for example:
- BSA acts as a stabilizing agent for many restriction enzymes
- Others cleave optimally at a temperature above the standard 37 °C
- Some enzymes aren’t very good at working overtime (or for longer than 1-2 hours)
- Most restriction enzymes digest efficiently between pH 7.2 and pH 8.5, so make sure to use the right buffer!
Always play it safe and check with the enzyme manufacturer for specific guidelines on your enzyme of choice. Most commercial suppliers have their own special guide on choosing and using restriction enzymes.
In a double restriction digest, you may need to partially sacrifice the activity of one enzyme if optimal conditions are different for the two enzymes. Keep that in mind when analyzing your results. Alternatively, you could perform a sequential digest, where you digest with the first enzyme, gel extract to remove the buffer components, then digest with the second enzyme. If you go down this route, it may be wise to start out with multiple tubes of the same reaction, as you will likely lose a lot of material during the gel extraction.
Lastly, you don’t want to overload your digest reaction when you run your gel. Agarose gels are not the most sensitive, and depending on your detection method, you can generally detect from as little as a few ngs (50-100 ng if using ethidium bromide) to a microgram. Although you can load more, it is not wise to try to visualize more than 1 µg/lane as you may find it difficult to distinguish the digested bands.
2. Methylation: Angel & Devil
When bacteria replicate a plasmid, they often methylate specific CpG islands. These are sequences that are often targeted for methylation. Methlyation may block a site from restriction enzyme cleavage. This may give you a headache if the enzyme you wish to use is methylation-sensitive – i.e. it cannot bind recognition sites that are methylated.
There are three different types of methylation enzymes typically found in laboratory E. coli strains: Dam methylase, Dcm methyltransferase and EcoKI methylase. If you are trying to digest a restriction site that may be methylated, you can use a methylation-incompetent strain of E. coli (e.g. JM11) to propagate your plasmid. These E. coli strains are incapable of methylating DNA, thus allowing your restriction enzyme to cleave CpG islands. Remember that deleted (denoted with ?) or disrupted genes are noted as part of strain genotypes. This will help you find strains with the right methylation status.
On the other hand, methylation of plasmids can be exploited as part of an experimental setup. For example, during site-directed mutagenesis, which uses PCR and cloning to mutate part of a DNA sequence, we can exploit the fact that plasmid DNA will be methylated, whereas the PCR product (if successfully amplified) will not be. Thus, protocols often include a DpnI digest step to remove the template plasmid DNA, leaving only the desired PCR product ready for ligating back into a digested plasmid.
3. Keeping the Stars Away: Minimizing Star Activity
Restriction enzymes actually have a dark side! In the right conditions, they can become promiscuous and digest DNA randomly, rather than at their specific recognition sites. This phenomenon is called star activity and is generally caused by long incubation periods (check guidelines for your enzyme) or suboptimal buffer conditions (e.g. pH). Therefore, it is critical to use your desired enzyme with the recommended buffer!
In addition, high glycerol concentration may lead to an in increase star activity. Since most enzymes and their buffers come packaged in glycerol to extend shelf life, you should adequately dilute both the buffer and enzyme. This is why many companies suggest a 20 – 50 µl reaction in their general protocols and provide the buffer as a 10x mix.
4. Cutting It Close – Give Your Enzymes Some Space
Always remember that some enzymes are most efficient when they have several base pairs on either side of the recognition site. This is particularly important if you are doing a double digest with the two enzymes very close together or if digesting the ends of a PCR product.
Each manufacturer has specific suggestions for their products, but it is often recommended to ensure that there are at least six base pairs on either side of the recognition site. The easiest way to add more base pairs is to engineer them via your PCR primers. Just try to avoid sequences carry a risk of primer dimer formation!
5. The Useful Cousins: Isochizomers and Neoschizomers
Sometimes you need to use a specific enzyme whose optimal conditions simply don’t suit your experimental setup. In this situation, isochizomers and neoschizomers may aid you in your experiment without seriously influencing your end result.
Isochizomers and Neoschizomers
An isochizomer is a restriction enzyme that recognizes the same site as another restriction enzyme but has different properties. For example, both SinI and AvaII cleave the sequence: G/G(A or T)CC. However, AvaII is methylation-sensitive, whereas SinI is not. Therefore, if you wanted to cleave the above sequence and don’t want to use a methylation-insensitive E. coli strain, SinI would suffice, whereas AvaII would not.
Two enzymes that share the same recognition site but cleave at a different base pair (bp) are termed neoschizomers. These may be useful in avoiding star activity and/or methylation sensitivity. For example, both KpnI and Acc65I recognize the site: GGTACC but cut in different positions. KpnI cuts after bp 5: GGTAC/C whereas Acc65I cuts after bp 1: G/GTACC. KpnI may exhibit star activity, whereas Acc65I is more robust. Thus, if it doesn’t matter where you cut, Acc65I may be a better choice.
Both isochizomers and neoschizomers may be useful in resolving tricky digests as they can allow you to circumvent star activity. And issues with methylation status and enzyme activity, often leaving you with the same or similar end product.
While restriction enzymes have helped us map genes, identify plasmids and clone to our hearts content for over 40 years, they require some planning! Hopefully these five tips will help you with your restriction digest needs. And keep these enzymes working for another 40 years!