Restriction enzyme overview
- General information about restriction enzymes
- Star activity of restriction enzymes
- Inactivation of restriction enzymes
- Buffer activity with restriction enzymes
- Universal buffers for double digestion with restriction enzymes
- Restriction enzymes affected by methylation
- Methylation-sensitive restriction enzymes
- QC of restriction enzymes
- Ligation cloning overview
- Ligation product guide
General information about restriction enzymes
Our restriction enzymes are classified into three categories, according to their cofactor requirements and the characteristics of their cleavage sites:
|Type||Cofactor(s)||Cleavage site||Example enzymes|
|Type I||ATP, AdoMet, Mg2+||Recognition sites and cleavage sites are different; cleavage sites are not fixed||EcoB, EcoK|
|Type II||Mg2+||These enzymes cleave DNA at the recognition sequence or at a defined distance from the recognition site||EcoRI, BamHI|
|Type III||ATP, Mg2+||These enzymes cleave DNA at fixed sites, although their recognition sites and cleavage sites are different||EcoPI, HinfIII|
Type II restriction enzymes are generally used in genetic engineering experiments. Restriction enzymes induce cleavage and/or star activity, depending on the substrate DNA and reaction conditions. Close attention should be paid to the factors described below.
DNA methylation effects
DNA prepared from host bacteria that express DNA methylase are partially methylated, and due to this methylation, they are fairly resistant to restriction enzymes that recognize and cleave methylated sites. Sites undergoing methylation vary depending on substrate DNA and host bacteria species. For example, in the case of E. coli, the following reactions occur depending on host species.
E. coli strains commonly used in transformation such as C600, HB101, and JM109 contain both dam and dcm methylases. Thus attention should be paid when DNAs prepared from these strains are used. In addition, most of the CG sequences in DNA derived from animals are modified to 5mCG, while the CG and CNG sequences in DNA derived from plants are generally modified to 5mCG and 5mCNG, respectively.
For additional information, please refer to our page about the effects of methylation on restriction enzyme activity.
Under certain reaction conditions, a restriction enzymes can lose its specificity and cleave base sequences that are different from the original recognition sites. This phenomenon is called star activity, and while almost all restriction enzymes have star activity, its occurrence depends on the enzyme, substrate DNA, and reaction conditions. In addition to this relaxation of recognition sites, nicking activity—in which substrate DNA is partially cleaved—is also observed. In order to suppress star activity, we recommend setting up restriction digests at lower glycerol concentrations, neutral pH, and higher salt concentrations. Note, though, that these conditions may also create lower reactivity overall.
For additional information, please refer to our page about compensating for star activity.
In cases where sufficient cleavage is not obtained with a restriction enzyme that should react with substrate DNA, several factors may be the cause. In addition to the methylation effects and star activities mentioned above, reduced enzyme activity, DNA purity, reaction inhibitors, or substrate DNA species may be responsible. In particular, DNAs with different sizes or site numbers require differing amounts of enzymes for complete cleavage, and therefore the calculated amount of enzyme required differs from the actual amount needed for an experiment. This difference is believed to stem from the interaction between the enzyme and the higher structure around its recognition site. For example, NaeI exhibits highly resistant cleavage sites in pBR322 DNA. Furthermore, a change in the content of a reaction mixture, e.g., addition of spermidine (Conrad and Topal, 1989) can change the cleavage order.
Conrad, M. & Topal, M. D. DNA and spermidine provide a switch mechanism to regulate the activity of restriction enzyme NaeI. Proc. Natl. Acad. Sci. USA 86, 9707–9711 (1989).
DNA binding substances
In gel electrophoresis following restriction digestion, issues such as no observable bands, broad bands, or atypical migration of bands can arise. These issues are due in part to the formation of complexes between the DNA and the enzyme or contaminating proteins. Addition of a denaturant such as SDS can improve electrophoresis results.
Definition of activity
One unit of restriction enzyme activity is defined as the amount of enzyme required to produce a complete digest of 1 µg of λ DNA in 60 minutes at 37°C in a 50-µl reaction volume. Relative activities in universal buffers can be found on the buffer activity page: its relative activity table lists the buffers, substrates, and temperatures used to determine enzyme activity, as well as the recommended buffers for each enzyme.
Each lot of enzyme is assayed based on the following experiments:
- Overdigestion test
1 µg of substrate DNA (usually λ DNA) and excess amounts of enzyme are incubated for 24 hours. Nonspecific DNase activity is measured using agarose gel electrophoresis.
- Genomic DNA analysis
20 to 150 units of restriction enzyme is added to the appropriate bacterial genomic DNA (agarose-embedded, 0.5 µg DNA/50 µl gel). After a 24-hour incubation, pulsed field electrophoresis is performed to confirm normal a DNA band pattern. Data is available on the restriction enzyme QC analysis page.
- Ligation and recutting test
Substrate DNA is digested with a 2- to 50-fold excess of enzyme. Digested DNAs are collected and dissolved in T4 DNA Ligase buffer (66 mM Tris-HCl [pH7.6], 6.6 mM MgCl2, 10 mM DTT, 0.4 mM ATP) to obtain a 0.1 to 1.0 µM concentration of 5'-terminus. An appropriate amount of T4 DNA Ligase is added and incubated for 1 hour or for 16–18 hours at 16°C. Collected DNAs are dissolved in the restriction enzyme reaction mixture and recut by the same enzyme. Contamination with ligase inhibitor, phosphatase, and exonuclease is determined from test results. Data is available on the restriction enzyme QC analysis page.
- pKF3 cloning test
This test is performed on restriction enzymes that have cleavage sites within the multiple cloning site in pKF3 DNA. pKF3 DNA is incubated with a 10-fold excess of restriction enzyme. After an inactivation procedure, digested DNAs are incubated using DNA Ligation Kit, Version 1 for 30 minutes at 16°C. TH2 competent cells are transformed with a portion of the reaction mixture and cultured on two kinds of plates (LB-Cm-Sm, LB-Cm) for 2 days at 37°C. Extremely small amounts of exonuclease are indicated by the appearance of colonies on the LB-Cm-Sm plates. Data is available on the restriction enzyme QC analysis page.
Residual activity after inactivation procedures
For each enzyme, residual activity is measured following these four inactivation procedures. Results can be found on the restriction enzyme inactivation page.
- Heating at 60°C for 15 minutes
- Heating at 70°C for 15 minutes
- Ethanol precipitation
- Phenol extraction
Store each enzyme at -20°C. The enzyme solution is not frozen at -20°C. Each enzyme is not inactivated by one or two cycles of freeze-thaw procedure, but do not repeat freeze-thaw cycles. (An exception is FseI, which is stored at -80°C.)
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