Restriction Enzyme Digestion: Key Considerations

The considerations for complete digestion can be grouped under two key themes: general best practices and steps to minimize specific challenges.

By conventional definition, one unit of restriction enzyme is required to completely digest 1 μg of specific DNA substrate such as Thermo Scientific pUC19 vector in a 50 μL reaction within 1 hour under optimal conditions. While the unit definition provides a standard measure, various DNA substrates may require different conditions for optimal digestion. Frequency of the recognition sequence and type of DNA influence optimal reaction conditions. Typically, enzyme suppliers recommend a 5- to 20-fold excess of enzyme (or 1 μL of enzyme per digestion reaction) for complete digestion, due to potential variations in quality, quantity, and the nature of DNA samples.

To help reduce these optimization steps, some restriction enzymes are designed with a single buffer, regardless of the type of DNA substrates or the number of restriction enzymes used.

To help ensure efficient activity of restriction endonucleases, they should be properly stored and used before their expiration date. In general, enzymes should be kept at –20⁰C in a non-frost-free freezer, preferably in small aliquots, to maintain activity and to minimize freeze-thaw cycles.

DNA samples should be free of contaminants such as nucleases, salts, organic solvents such as phenol, chloroform, or alcohol, and detergents that can inhibit enzymes and cause other adverse effects.

Restriction enzymes are often supplied in 50% glycerol to prevent freezing at –20°C. However, the viscosity of glycerol may make pipetting and dispensing small volumes of enzyme during reaction setup difficult. It is best to add enzymes last to complete the final volume, and to mix well upon addition. “Flicking” to gently mix the reaction tube contents, and then briefly spinning the tube, will help to disperse the enzyme throughout the reaction mixture and get the reaction solution collected at the bottom of the tube.

During incubation, reactions must reach the desired temperature, which should remain constant over the incubation period. This is especially crucial when “fast” restriction enzymes, which are designed to complete a digestion in 5–15 minutes, are used in place of “conventional” enzymes with 1-hour incubation times. Special attention should be given to sealing the reaction tubes to avoid sample evaporation, which can occur with prolonged incubations (e.g., >1 hour) and small volumes (e.g., <10 μL).

When choosing restriction enzymes, one important aspect that is often overlooked is manufacturing practices and quality processes of the suppliers. To obtain reliable results and minimize variations between experimental runs, researchers should be aware of quality control and quality assurance implemented by enzyme suppliers in producing their products.

Star or “relaxed” activity is an inherent property of restriction endonucleases where, under non-optimal conditions, a restriction enzyme may act on recognition sequences with minor differences from their canonical recognition sites. For example, as illustrated in Figure 1, EcoRI recognizes and cleaves the site 5’-GAATTC-3’, but its star activity may result in cleavage at 5’-TAATTC-3’ and 5’-CAATTC-3’. Similarly, BamHI may cut 5’-NGATCC-3’, 5’-GPuATCC-3’, and 5’-GGNTCC-3’ (where N stands for any nucleotide) in addition to its normal recognition sequence, 5’-GGATCC-3’.

Under optimal reaction conditions, the rate of cleavage at the “star” site(s) is much lower than at the normal recognition site (approximately 10⁵–10⁶ difference). This rate can significantly Increase under specific conditions:

• Excess amounts of restriction enzymes relative to the amount of DNA can result in star activity, as the enzyme may begin to recognize and cut at similar but non-specific sites.
• Prolonged incubation times (over-digestion) beyond the recommended duration can increase the likelihood of star activity, as the enzyme may begin to cut at non-specific sites over time.
• Higher glycerol percentages (e.g., >5%) often present in the enzyme storage buffer can induce star activity.
• Suboptimal salt concentrations, particularly low ionic strength, can affect the enzyme's specificity and lead to star activity.
• Organic solvents such as ethanol, dimethyl sulfoxide (DMSO), or isopropanol can affect the enzyme's specificity and promote star activity.
• Deviations from the optimal pH for the enzyme's activity can result in reduced specificity.
• Divalent cations other than Mg²⁺ can contribute to nonspecific cleavage of the substrate DNA.

Some restriction enzymes are designed to avoid star activity by operating in a single optimal buffer system and rapidly digesting target DNA in 5–15 minutes, maintaining high specificity throughout the process.

Star activity can be prevented by using the protocol and buffers recommended by the enzyme manufacturer.

Some restriction enzymes,when used in conjunction with their buffers, have been optimized to show no star activity even after overnight digestion.

Incomplete digestion occurs when restriction enzymes do not fully cut all the recognition sites in a DNA sample. This results in a mixture of fully digested, partially digested, and undigested DNA fragments. Incomplete digestion can compromise the accuracy and efficiency of downstream experiments.

Incomplete digestion with a restriction enzyme may occur when too much or too little enzyme is used. The presence of contaminants in the DNA sample can inhibit the enzymes, also resulting in incomplete digestion. Suboptimal reaction conditions such as buffer composition, incubation time, and reaction temperature are also common causes of incomplete digestion.

Some restriction enzymes require cofactors for full activity. For instance, Esp3I (BsmBI) requires DTT, while Eco57I (AcuI) needs S-adenosylmethionine. Some restriction enzymes such as AarI and BveI (BspMI) require two copies of the recognition site for efficient cleavage; for these restriction enzymes, an oligonucleotide with the recognition site is often added to the reaction to enhance enzymatic activity.

In addition to reaction conditions and enzyme properties, the nature of the DNA may play a role in the restriction digestion (Table 1). Methylated DNA can be resistant to cleavage by some restriction enzymes (see the methylation section for details). Proximity of enzyme recognition sites to the termini (in linear substrate DNA), as well as proximity between cleavage sites (in double digestion) may determine how efficiently enzymes cut the DNA. Furthermore, some supercoiled DNA molecules are more challenging to cleave than their linear counterparts and increasing the amount of enzyme 5- to 10-fold can help with complete digestion.

To achieve complete digestion, closely follow protocols and recommendations provided by manufacturers.

Table 1. Common causes of incomplete digestion.

Check out more troubleshooting tips for unexpected cleavage patterns

Unexpected cleavage patterns are characterized by DNA fragments that differ from the anticipated fragment size after restriction digestion and can be result of star activity, incomplete digestion, and mutations.

Both star activity and incomplete digestion give rise to unexpected patterns on the gel. Therefore, it is essential to differentiate the two for troubleshooting.

Another cause of unexpected cleavage is mutations that may be introduced into the substrate DNA during propagation and amplification. A mutation may destroy a known restriction site, preventing the restriction enzyme from recognizing and cutting at that specific location, or it may create a new restriction site, leading to additional, unintended cuts. This can produce DNA fragments that differ from the anticipated sizes, complicating the interpretation of results. To identify whether mutations are the cause of the unexpected cleavage Sanger sequencing of the DNA can be employed as it is a useful method to determine whether a mutation is the cause of unexpected cleavage of the substrate DNA. Once identified, strategies such as using different restriction enzymes, correcting the mutations, or redesigning primers for amplification can be employed to mitigate the effects and achieve the desired DNA cleavage.

To minimize unexpected cleavage patterns, pay careful attention to experimental setup and execution:

• Optimize reaction conditions—Follow the recommended incubation time, enzyme concentrations, and temperature to avoid over-digestion or incomplete digestion.
• Select optimal buffer—Use available online tools to select optimal buffer when performing double digests to ensure that both enzymes are compatible with the same buffer or use a sequential digestion strategy if necessary. Alternatively, use enzymes developed to work in a single universal buffer.
• Check for DNA methylation—Some restriction enzymes are sensitive to methylation at their recognition sites, which can inhibit cleavage. Use a methylation-insensitive enzyme or a bacterial strain that does not methylate the DNA.
• Dissociate enzyme-DNA complexes—Using a loading dye containing SDS, and heating to dissociate the enzymes from the cleaved DNA, can prevent such gel shifts when analyzing your digestion reactions.
• Verify DNA sequence—Sequence the DNA to confirm that the expected restriction sites are present and have not been altered by mutations.
• Proper handling and DNA quality—It is imperative that the restriction enzyme, DNA, and other reagents used in the reactions are free of contaminants such as another restriction enzyme, non-target DNA, nucleases, salts or organic solvents.
• Storage conditions—Store restriction enzymes at the recommended temperature and minimize freeze-thaw cycles to prevent degradation and loss of activity and maintain their specificity.

Sensitivity of restriction enzymes towards methylated DNA recognition sites depends on the restriction enzymes. For example, as illustrated in Figure 4, Dam methylation at GATC completely blocks MboI but activates DpnI. On the other hand, CpG methylation at 5′-CCGG-3′ blocks HpaII activity but has no effect on MspI. In some cases, restriction enzyme activity is only partially inhibited by methylation (e.g., XhoI).

When propagating plasmids in bacteria, the effects of methylation on restriction enzymes of interest must be considered. To prevent methylation at 5′-GATC-3′ and 5′-CCWGG-3′, competent cells that lack Dam and Dcm methylases (dam–/dcm–) should be chosen for plasmid transformation. Most E. coli cells do not possess a CpG methylation system, so CpG methylation is not a concern for DNA isolated from bacteria. If genomic DNA is extracted from plants and mammals, methylation may occur at the CpG sites and impact direct restriction digestion by some methylation-sensitive enzymes.

Read more on digestion of methylated DNA