Detection of specific sequences among DNA fragments separated by gel electrophoresis. Journal of Molecular Biology 98 , — Restriction Enzymes.
Genetic Mutation. Functions and Utility of Alu Jumping Genes. Transposons: The Jumping Genes. DNA Transcription. What is a Gene? Colinearity and Transcription Units. Copy Number Variation. Copy Number Variation and Genetic Disease. Copy Number Variation and Human Disease. Tandem Repeats and Morphological Variation. Chemical Structure of RNA. Eukaryotic Genome Complexity. RNA Functions. Restriction Enzymes By: Leslie A. Pray, Ph. Citation: Pray, L. Nature Education 1 1 Restriction enzymes are one of the most important tools in the recombinant DNA technology toolbox.
But how were these enzymes discovered? And what makes them so useful? Aa Aa Aa. When I come to the laboratory of my father, I usually see some plates lying on the tables.
These plates contain colonies of bacteria. These colonies remind me of a city with many inhabitants. In each bacterium there is a king.
He is very long, but skinny. The king has many servants. These are thick and short, almost like balls. My father calls the king DNA , and the servants enzymes. My father has discovered a servant who serves as a pair of scissors. If a foreign king invades a bacterium, this servant can cut him in small fragments, but he does not do any harm to his own king. Initial Steps in Restriction Enzyme Research.
Figure 1. Figure Detail. Learning to Use Restriction Enzymes. Cutting with Restriction Enzymes. This article has been posted to your Facebook page via Scitable LearnCast. Change LearnCast Settings. Scitable Chat. Register Sign In. After binding at a non-cognate sequence, several enzymes have been shown to locate their targets through linear diffusion.
During this process a large number of water molecules appear to fill the spaces between the enzyme and the DNA. Once the cognate recognition sequence is found, much of the water is excluded as a highly redundant number of contacts evolve between the enzyme and the bases and phosphodiester backbone of the DNA. In the case of EcoRI, 50 water molecules are excluded at the cognate site 6.
Generally, non-specific bases on either side of the target sequence are required for proper recognition. Conformational changes occur in both the enzyme and DNA as the specific complex forms. The resulting induced fit positions the catalytic center in reactive proximity to the substrate. Using the known co-crystal structures of enzymes bound to their cognate sequences and substitution experiments in the enzyme or DNA for a limited number of additional enzymes, a mechanism for DNA cleavage has been postulated.
Evidence for most enzymes studied to date supports a substrate assisted catalysis model 7. Hydrolysis begins by in-line nucleophilic attack of an activated water molecule. Although all restriction enzymes bind DNA nonspecifically, under optimal conditions the difference in cleavage rates at the cognate site and the next best site single base substitution is very high.
However, under non-optimal conditions, the differences in cleavage rates between cognate and next-best sites change dramatically for many enzymes. This loss of fidelity or increase in cleavage at sites similar to the cognate site is commonly referred to as star activity. A number of reaction parameters can increase the rate of cleavage at star sites relative to cognate sites.
In conjunction with this increase in star activity, cleavage rates at the cognate site generally decrease. Several plausible explanations for star activity are based on the proposed mechanisms for target site identification and hydrolysis see Structure and Mechanism of Action for more information. During nonspecific binding, a large number of water molecules are present at the protein-DNA interface.
When tighter binding and positioning of the catalytic site occurs upon recognition of the target sequence, the number of these interface water molecules is significantly reduced. The higher osmotic pressure caused by volume excluders results in the same reduction in the amount of interface water molecules and allows easier active complex formation at star sites 3. At alkaline pH, higher OH - concentrations may reduce the need for an activated water molecule, which normally initiates nucleophilic attack on the scissile phosphorous.
Although all restriction enzymes probably exhibit some decrease in the cleavage rate difference between cognate and near-cognate sites under such extreme conditions as 4M ethylene glycol, most are not significantly affected under common usage conditions. Those that are susceptible to star activity are induced to different degrees by variations in reaction conditions or by combinations of the conditions listed above.
The Table below lists the enzymes sold by Promega that may exhibit star activity, especially under reaction conditions that deviate from those recommended. In multiple enzyme digests or multiple step applications, it is advisable to stay at or near the optimal conditions for these enzymes whenever possible. When presented with multiple recognition sites that differ in their flanking sequences, most restriction enzymes exhibit slight preferences and cleave the sites at different rates.
These rate differences are such that the addition of a small excess of enzyme will avoid any problems due to incomplete digestion. As always, however, one must be aware of the experimental molar concentration of recognition sites and digest conditions relative to that of the unit definition.
See Substrate Considerations for further information. A few restriction enzymes have considerably greater difficulty in cleaving some of their recognition sites.
Original experiments with these enzymes led to designation of their site preferences as shown:. Enzymes that have cleavable, slow, and resistant sites in the same or different DNAs have been designated Type IIe restriction enzymes.
There is evidence to suggest that Eco57I also belongs to this group 2. Investigation revealed that binding of a second recognition sequence, in cis or trans , to a distal, non-catalytic site on the enzyme allows slow and resistant sites to become cleavable. This e ffector sequence alters the kinetics in one of two ways.
In the V class NaeI, BspMI , binding of activator DNA increases the V max without changing the K m , indicating that the increased catalytic activity is not related to the affinity of the enzyme for its substrate. It is assumed that the flanking sequences of a recognition site influence the kinetics of cleavage at that site, but at this time the interaction is not understood.
Considerable differences also exist in the ability of effector sequences to stimulate cleavage. Generally, a recognition site flanked by the sequence from a site that is cleaved easily is a useful starting point for designing good effector sequences.
Each restriction enzyme has optimal reaction assay conditions and different conditions for long term storage. The recommended assay and storage conditions are both determined by the manufacturer to provide the user with the highest activity, best fidelity and greatest stability for each enzyme.
Factors that must be considered include temperature, pH, enzyme cofactors, salt composition, ionic strength and stabilizers. Promega restriction enzyme Reaction Buffers are designed to provide the best balance of optimal activity and convenience. All enzyme storage conditions are validated through our Quality Assurance re-assay program to maximize long term stability.
Setting up digests with a single restriction enzyme is relatively straightforward. However, digests using multiple enzymes that have different buffer requirements may demand the use of alternative buffers and may require adjustments in the number of units of enzyme used. If no compatible buffer can be found a sequential reaction may be performed in which additional buffer or salt is added to the reaction before the second enzyme, or each digest may be performed sequentially using the optimal buffers.
The latter option will require either a DNA precipitation or purification step after the first digest. Regardless of the type of digest performed, the addition of BSA is recommended to stabilize the enzyme and enhance activity 1 2. Salt Concentration: Restriction enzymes are diverse in their response to ionic strength. A few enzymes prefer acetate to chloride anions. Suboptimal ionic strength or type of ion may lead to star activity. BSA: Bovine Serum Albumin is used in restriction enzyme storage buffers and is added to digestion reactions to stabilize the enzyme.
BSA can protect restriction enzymes from proteases, non- specific adsorption and harmful environmental factors such as heat, surface tension and interfering substances. Typically, the addition of 0. The Acetylated BSA provided with Promega's restriction enzymes has been modified and extensively tested to ensure that no degrading activities are present. A few enzymes require higher or lower temperatures for optimal activity e. For incubations greater than 1 hour with high temperature enzymes, cover the reactions with a drop of mineral oil to prevent evaporation.
Generally, the incubation temperature for the enzyme reflects the growth temperature of the bacterial strain from which it is derived. This type of information is particularly useful when performing double digests. Volume: Viscous DNA solutions inhibit enzyme diffusion and can reduce enzyme activity. DNA concentrations that are too dilute can fall below the K m of the restriction enzyme and also affect enzyme activity.
Use of an unusually large volume of DNA or enzyme may give aberrant results. Caution should be exercised to prevent higher than normal concentrations of EDTA and glycerol. The following is an example of a typical analytical single restriction enzyme digestion:. Larger scale restriction enzyme digestions can be accomplished by scaling this basic reaction proportionately. If all of the restriction enzymes in a multiple digest have the same optimal buffer, setting up the digest is straightforward.
However, when this is not the case, several options are available. Note: Perform each digest sequentially using the optimal buffers. This will require either a DNA precipitation or purification step after the first digest. Although this procedure involves more steps than those listed above, in situations where options are not satisfactory, it may be the best alternative. Some common controls used for restriction enzyme digestion and gel analysis are given in the Table below. Restriction enzymes differ in their reaction kinetics.
Variations in the number of enzyme units used and the reaction incubation times were tested. Incubation time for the unit definition assay is one hour. The concentration of the DNA sample can influence the success of a restriction digestion.
Viscous DNA solutions, resulting from large amounts of DNA in too small of a volume, can inhibit diffusion and can significantly reduce enzyme activity 1. DNA concentrations that are too low also may inhibit enzyme activity see Substrate Quality.
Typical K m values for restriction enzymes are between 1nM and 10nM, and are template-dependent 2. Recommended final DNA concentrations for digestion range from 0. Substrate structural variations, concentration and special considerations are discussed below according to DNA type. In lambda DNA, the cos ends, base, complementary, single-stranded overhangs at the end of each molecule may re-anneal during digestion.
This can give the appearance that digestion is incomplete. Compared to linear DNA, plasmids often require more units of restriction enzyme for complete cleavage due to the supercoiling 1 or the total number of sites to be digested see Recognition Site Density.
See Digestion of Supercoiled Plasmid DNA for information on the relative units needed for complete cleavage of a typical plasmid vector with common cloning enzymes.
If a supercoiled plasmid is first linearized with another restriction enzyme or relaxed with topoisomerase, less enzyme may be needed for digestion. Viscosity can be adjusted by increasing the reaction volume.
Addition of spermidine to final concentration of mM also has been reported to increase enzyme activity in the digestion of genomic DNA 4. Addition of BSA to restriction digests at a final concentration of 0. The number of enzyme units needed must be balanced with the total number of sites to assure complete cleavage.
Longer incubation times may be required to ensure complete digestion. Consult the Promega Product Information sheet for the overdigestion value of the enzyme. For many common restriction enzymes, acceptable activity is seen in PCR buffer, although digestion after amplification may not result in the expected compatible ends due to residual polymerase activity 5. Digestion near the end of a PCR product may also present problems. If an oligonucleotide primer is designed with a cut site that is too close to the end of the DNA, the site may cut poorly or not at all.
Since it is very difficult to assay for cutting near the end of DNA, the effectiveness of compensation with extra enzyme units or increased incubation time is difficult to determine. Another reason for incomplete digestion of PCR fragments may be primer dimers. If the restriction site is built into the primer, primer dimers will contain a double-stranded version of the site, usually in vast molar excess over that of the desired target PCR fragment. Double-Stranded Oligonucleotides: Many of the same considerations for PCR products apply to the digestion of double-stranded oligonucleotides.
In this case high densities of recognition sites per unit of mass can be present and the site may also be near the end of the DNA molecule. Studies have shown, however, that several restriction enzymes that appear to cleave single-stranded DNA actually recognize folded-back duplex regions within the single-stranded genomes e.
Therefore, these enzymes are not digesting single-stranded DNA, rather individual sites that are in the duplex form. Digestion required 20 to fold higher enzyme levels than those needed for duplex DNA. It is possible but not proven that the RNA was also cleaved with large excesses of enzyme. Influence of Flanking Sequence: The sequences flanking the restriction enzyme recognition sequence can influence the cleavage rate of many restriction enzymes although the differences are usually less than fold.
A small number of enzymes e. Methylation: Methylation of nucleotides within restriction enzyme recognition sequences can affect digestion. Methylation may occur as 4-methylcytosine, 5-methylcytosine, 5-hydroxymethylcytosine or 6-methyladenine in DNA from bacteria including plasmids , eukaryotes and their viruses.
The sensitivity, or lack thereof, to site-specific methylation, is known for many restriction enzymes Often, isoschizomers differ in their methylation sensitivity. Refer to Cat. A provide an easy and effective way to isolate and purify DNA, free of salt or macromolecular contaminants. Genomic DNA purified by traditional techniques can contain double-stranded breaks due to mechanical shear forces.
Such breaks can be a source of background in megabase mapping of fragments of kb. To avoid this, mammalian, bacterial and yeast cells can be embedded in agarose strips and the cells lysed and treated with proteinase K in situ Most restriction enzymes can cut DNA embedded in agarose provided that more enzyme and longer incubation times are used.
The anti-coagulant used during blood collection can affect the ability of restriction enzymes to completely digest DNA. Use EDTA as an anti-coagulant rather than Heparin, which can bind tightly to the enzyme and interfere with digestion.
A number of rapid DNA purification protocols have been written that do not require separation of white cells from red cells 12 These techniques can yield good quality DNA from small volumes of blood, but the DNA obtained after scale-up may be of poorer quality.
DNA purified with this system is suitable for digestion with restriction enzymes. A provides a reliable method for purification of double-stranded PCR-amplified DNA from any salts or macromolecular contaminants. When digesting other substrates, adjustments may be needed based on the amount of substrate, the number of recognition sites per molecule and the incubation time. The following table illustrates the effect of differences in substrate recognition sites per molecule for EcoRI while keeping the substrate mass and incubation time constant.
A 2 3 or by embedding the cells of interest in blocks or beads of agarose and enzymatically digesting the cell membranes and proteins 4. Large DNA is quite susceptible to mechanical shearing and it is difficult to obtain DNA of 50kb or more unless it is embedded in agarose. Regardless of the preparation method, genomic DNA is frequently less pure than plasmid or other smaller DNA that can be treated more harshly during isolation.
In addition, genomic DNA, especially that of higher organisms, may contain more modifications such as methylation. The methylation sensitivity of potential restriction enzymes may need to be considered for genomic digests. Excess restriction enzyme units and extended incubation times are standard for genomic digestions.
For long incubations, especially at elevated temperatures, evaporation of water from the buffer can concentrate components of the reaction and cause star activity. The reaction can be overlaid with mineral oil or the digestion performed in an incubator to avoid evaporation.
Addition of spermidine to a final concentration of mM has also been shown to be helpful for genomic digests 1 5. Incubation time is typically 4 hours to overnight. A general protocol for embedding and digesting mammalian cells in agarose is provided below. Conditions will differ significantly for other cell types. The conditions required for digestion of agarose-embedded DNA differ from those required for digestion of DNA in solution.
In general, much more restriction enzyme is needed. We have tested a number of enzymes for their ability to digest DNA embedded in agarose see Table below. The exact amount of enzyme needed varies depending on the DNA type and preparation.
A general protocol for digestion of agarose embedded DNA is provided below. It is possible to calculate the expected average fragment size for a given genomic DNA if the percent GC content of the DNA and the recognition sequence of the restriction enzyme are known. The probability of cutting any given 6 base sequence is 0. The equation can be refined if there is a known bias in the frequency of dinucleotide and trinucleotide repeats in the DNA being digested 5.
For a sequence N 1 N 2 N 3 N 4 N 5 N 6 where N 1 through N 6 are the bases in the restriction enzyme recognition sequence , the expected frequency of digestion can be calculated as. Where p N is the frequency of N in the genome and p N a N b is the dinucleotide repeat frequency. Where p N a N b is the dinucleotide repeat frequency and p N a N b N c is the trinucleotide repeat frequency.
The GC content and dinucleotide frequencies of many organisms have been determined 6. Because the sequences of many organisms have been elucidated it is now possible to generate complete restriction maps of entire genomes. Larger DNAs re-orient more slowly and thus have slower net migration rates. Restriction enzyme units are usually defined using linear DNA substrates containing multiple recognition sites as these tend to give more reproducible results. Lambda and Adenovirus are the two substrates used most frequently because of their commercial availability and high quality.
Molecular biology applications frequently involve cutting a supercoiled plasmid at a single site within the multiple cloning sequence. There are several reasons why this is the case. For example, there are 0. HindIII cleaves this substrate 7 times or 0. For a 3, base pair plasmid with a single recognition site, there are 0. The ability of a restriction enzyme to find a single site by linear diffusion in the supercoiled plasmid is also presumed to be different than for any of the sites on a linear substrate.
Although it is not common, some enzymes exhibit differences in their ability to cut supercoiled DNA depending on the buffer conditions used. For example, SacII exhibits a pronounced difference in its ability to cut supercoiled plasmids depending on buffer conditions, but this sensitivity is not seen nearly as dramatically with linear substrates. In order to recognize and cleave their recognition sequence, most restriction enzymes need some flanking DNA.
Because of this it can be difficult to achieve complete digestion of PCR products that have restriction sites engineered near the end of a primer or to perform double digests using two enzymes that cut at sites close to each other in a polylinker region. Such digestions may be improved by using long hour incubation times. Methylation of DNA.
The next step is the ligation of the insert into the linearized vector. This involves the formation of phosphodiester bonds between adjacent 5'-phosphate and 3'-hydroxyl residues, which can be catalyzed by two different ligases: E. The latter is the preferred enzyme because it can also join blunt-ended DNA fragments.
The efficiency of the ligation reaction depends on:.
0コメント