General restriction enzyme reaction conditions
When setting up restriction digestion reactions, it is important to follow recommendations by the enzyme suppliers for optimal reaction conditions. Important parameters to consider include substrate (DNA) and enzyme amounts, reaction volume, and incubation time.
By conventional definition, one unit of restriction enzyme cleaves 1 μg of a defined substrate (e.g., plasmid pUC19) to completion in 1 hour in 50 μL under optimal conditions. While the unit definition provides a form of measurement, it should be noted that various DNA substrates in the presence of the same amount of restriction enzyme might have different optimal requirements based on the frequency of the recognition sequence in the DNA substrate and the types of substrate used. Often in practice, the 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 ensure efficient activity of restriction endonucleases, they should be properly stored and used before their expiration date per supplier recommendations. 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 (e.g., 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).
In addition to general considerations, one should be aware of the following aspects of restriction digestion when performing experiments:
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’.
|Figure 1. Potential star or relaxed activity of two common restriction enzymes, EcoRI and BamHI.
Specifically, 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). As such, excess amounts of restriction enzymes and/or prolonged incubation times (over-digestion) are common causes of star activity during digestion. In addition, higher glycerol percentages (e.g., >5%), low salt concentration, suboptimal pH, presence of organic solvents, and divalent cations other than Mg2+ can contribute to nonspecific cleavage of the substrate DNA. By using the protocol and buffers recommended by the enzyme manufacturer, star activity can be avoided.
Incomplete digestion is a frequently encountered issue when using restriction endonucleases. Incomplete digestion 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). Close 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.
Table 1. Common causes of incomplete digestion.
||Substrate DNA conditions
- Too little enzyme
- Too much substrate
- Non-optimal pH
- Non-optimal temperature
- Short incubation time
- Recognition sequence requirements
- Site preference
- Restriction site proximity
Enzyme suppliers provide usage recommendations in product inserts, which must be followed closely to achieve complete digestion.
Unexpected cleavage pattern
Even after following the manufacturer’s specifications, unexpected cleavage of the substrate DNA may be observed. To avoid such pitfalls, it is imperative that the enzyme and DNA, as well as reagents used in the reactions, are free of contaminants (e.g., another restriction enzyme, DNA, and nucleases).
One 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 or create a new one, producing unexpected results. Sanger sequencing of the DNA is a useful method to determine whether a mutation is the cause of unexpected cleavage of the substrate DNA.
Sometimes an unexpected pattern is a result of detection rather than digestion. Some restriction enzymes (e.g., FokI, TauI) may bind tightly to the DNA, resulting in an apparent gel shift in electrophoresis. Using a loading dye containing SDS, and heating to dissociate the enzymes from the cleaved DNA, can prevent such gel shifts (Figure 2).
|Figure 2. Restriction enzyme binding to the DNA may result in bands or smears above the expected bands in electrophoresis.
Both star activity and incomplete digestion also give rise to unexpected patterns on the gel. Therefore, it is essential to differentiate the two for troubleshooting. One approach to differentiate them is to run an incubation time course. With longer incubation, unexpected bands typically become more distinct if they are due to star activity, but disappear if they are due to incomplete digestion. In addition, the size of the unexpected bands can be a differentiating factor. Some unexpected bands from star activity appear lower than predicted, whereas all of those from incomplete digestion are located above the smallest expected band (Figure 3).
|Figure 3. Distinguishing incomplete digestion and star activity.
DNA methylation is one of the most common types of DNA modification found in both eukaryotes and prokaryotes. In bacteria, it is part of the restriction-modification (R-M) defense system against phage infection (see Restriction enzyme basics), whereby host bacteria protect their own DNA against cleavage by their endogenous restriction enzymes. Methylation commonly occurs at cytosine (C) and adenine (A) bases, and predominantly forms 5-methylcytosine (m5C), N4-methylcytosine (m4C), and N6-methyladenine (m6A) derivatives.
DNA methyltransferases drive the methylation reaction by transferring a methyl group from a donor to the acceptor bases (e.g., A and C). The most common types of DNA methyltransferases found in laboratory strains of bacteria include:
- Dam: stands for deoxyadenosine methyltransferase and converts the sequence 5′-GATC-3′ to 5′-G(m6A)TC-3′
- Dcm: stands for deoxycytosine methyltransferase and converts the sequence 5′-CCWGG-3′ to 5′-C(m5C)WGG-3′ (where W is either A or T)
- EcoKI: stands for the Type I R-M system in the E. coli K12 strain and modifies adenosines in the sequence 5′-AAC(N)6GTGC-3′
- EcoBI: stands for the Type I R-M system in the E. coli B strain and modifies adenosines in the sequence 5′-TGA(N)8TGCT-3′
In mammalian and plant systems, CpG or CpNpG methylation is a common DNA modification with implications in biological processes, making it a major focus of epigenetic studies.
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).
|Figure 4. Varying sensitivity of restriction enzymes towards substrate DNA methylation.
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.
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. Some key questions to consider when purchasing the enzymes include:
- What is the history and experience of the manufacturer? Do they have a long-standing reputation and success in research and discovery of restriction enzymes? ─ Enzymes should come from a trusted source to obtain consistent results.
- How are the enzymes manufactured? Are the production sites certified to meet international quality standards such as ISO 9001 and clean-room environments? ─ It is crucial that enzymes be reliable, with minimal batch-to-batch variation.
- What are the capabilities of the manufacturers? Can they provide batch production and custom packaging for large-scale experiments? ─ Consistent batch and custom production are important for genome-wide or high-throughput studies.
- What are the specifications and tests performed on the enzymes? How are their activity, purity, and application determined? Are those tests sensitive enough to ensure they are right for your experiments? ─ The availability of quality control documentation is a key aspect of quality assurance.
- What is the breadth and depth of knowledge offered by the suppliers? When there are questions about the enzymes or issues with the products, is prompt and helpful support available from the suppliers? ─ In-depth understanding and real-time support is a key for providers who function as partners.
In essence, not all restriction enzymes are created equal. The manufacturing practices, quality control, and quality assurance implemented to produce these invaluable and critical tools are important for the success of your experiments and thus are worth careful consideration.