Instructions for the Use of Restriction Enzymes
At present, the restriction enzymes discovered are classified into the following three types according to cofactor requirement and characteristics of cleavage sites.
|Type I||ATP, AdoMet, Mg2+||Their recognition sites and cleavage sites are different and cleavage sites are not fixed.||EcoB, EcoK|
|Type II||Mg2+||They cleave DNAs at the recognition sequence or at a defined distance from the recognition site.||EcoR I, BamH I|
|Type III||ATP, Mg2+||They cleave DNAs at fixed sites, though their recognition sites and cleavage sites are different.||EcoP I, Hinf III|
Among these restriction enzymes, type II enzymes are generally used in genetic engineering experiments. All the restriction enzymes currently available on the market are type II.
Restriction enzymes induce a wide variation of cleavage or star activity occurrence depending on substrates DNA and their reaction conditions. Thus, on the use of restriction enzymes, attention should be paid to these factors in order to obtain aimed cleavage. Instructions for the use of restriction enzymes are as follows.
1) Effects of DNA Methylation
DNAs prepared from host bacteria containing DNA methylase gene are partly methylated, and due to the methylation, they are fairly resistant to restriction enzymes which 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 CG sequences in DNAs derived from animals are modified to 5mCG, while CG and CNG sequences in DNAs derived from plants are generally modified to 5mCG and 5mCNG, respectively.
For further information, please refer to the table "The effect of methylation on restriction enzyme activity."
2) Star Activity
Some restriction enzymes lose their specificities to substrate DNA and cleave base sequences which are different from the original recognition sites in some reaction conditions. This phenomenon is called "star activity" of restriction enzymes. Almost all restriction enzymes have star activity, though their occurrences depend on enzymes, substrate DNAs and reaction conditions. Besides relaxation of recognition sites, partly cleaved DNA called "nicking activity" is also observed. In any case, in order to suppress star activity to the utmost, reactions at lower glycerol concentrations, neutral pH, and higher salt concentrations are recommended, though these conditions may cause lower reactivity. For further information, please refer to the table "The star activities of restriction enzymes."
3) Partial Cleavage
In the cases that sufficient cleavage is not obtained with restriction enzymes assumed to react on substrate DNA, several factors may account, including aforementioned 1) and 2), reduced enzyme activity, DNA purity, reaction inhibitors or substrate DNA species. In particular, DNAs with different size or site numbers require diverse amounts of enzymes for complete cleavage and thus calculated "amount of enzyme required to produce complete digestion of 1 µg of substrate DNA" and "actual amount" do not agree depending on enzymes. These differences are presumed to stem from the interaction between enzymes and higher structure around their recognition sites. In fact, Nae I exhibits highly cleavage resistant sites (Site Preference) in pBR322 DNA. Furthermore, a change in content of reaction mixture (addition of spermidine1) may change cleaving order.
1"DNA and spermidine provide a switch mechanism to regulate the activity of restriction enzyme Nae I," Conrad, M. and Topal, M.D. (1989) Proc. Natl. Acad. Sci. USA 86, 9707-9711.
4) DNA Binding Substances
In the electrophoresis after restriction enzyme reaction, troubles such as no observable bands, broad bands, and atypical migration of bands may occur. These troubles are probably caused by following reasons: DNA and enzyme itself or contaminated proteins form complexes, which do not enter into gel or undergo staining by ethidium bromide. In these cases, addition of denaturants such as SDS into samples to final concentrations around 0.1% might improve the electrophoresis result.
While stable durations for restriction enzyme activity are basically defined by the test as more than one year later, most of enzymes do not lose their activity rapidly after the term. Thus there is a possibility that even long-term stored enzymes can be used for experiments. Regarding to these stored enzymes, reassay of their titer before usage is recommended. Most of enzymes do not lose their activity in a frozen storage.
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 λDNA in 60 minutes at 37°C in a 50 µl reaction volume. Substrate and temperature used in the activity determination are described for each enzyme. Relative activities in universal buffers are summarized in the table "Buffer activity chart for restriction enzymes." In the relative activity table, buffers used in the activity determination and recommended ones are indicated in pink cells.
Each lot of every enzyme is assayed on the following headings.
- Overdigestion test
1 µg of substrate DNA (usually λDNA) and excessive amounts of enzyme are incubated for 24 hours. Then, nonspecific DNase is checked by agarose gel electrophoresis.
- Genome DNA analysis
Tests are performed on selected restriction enzymes (indicated for particular enzymes).
20 to 150 units of restriction enzymes are added to the appropriate bacterial genome DNA (Agarose-embedded, 0.5 µg DNA/50 µl gel). After 24 hours incubation, pulsed field electrophoresis is performed to confirm normal DNA bands pattern. Data are summarized in the table "Quality Control test using genomic DNA."
- Ligation-Recutting test
Substrate DNA is digested with a 2 to 50-fold excess of enzyme. Digested DNAs were 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 are determined from the test results. For more information on this assay, see Quality Control Test -- Ligation-Recutting.
- pKF3 cloning test
Tests are performed on the restriction enzymes which have a cleavage site at multi cloning sites in the enforcement cloning pKF3 DNA. pKF3 DNA is incubated with a 10-fold excess of restriction enzyme. After a inactivation procedure, digested DNAs are incubated using a DNA Ligation Kit, Ver. 1 (Cat.# 6021) for 30 minutes at 16°C. TH2 competent cells are transformed by 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 is judged from the appearance of colonies on the LB-Cm-Sm plates. For more information on this assay, see Quality Control Test--pKF3 Cloning.
Residual Activity after Various Inactivating Procedures
For each enzyme, residual activity was measured after the following 4 different inactivating procedures to examine the complete inactivating conditions. The results are summarized in the table "Residual activities following various inactivation treatments."
- Heating at 60°C for 15 minutes
- Heating at 70°C for 15 minutes
- Ethanol precipitation
- Phenol treatment-ethanol precipitation
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 Fse I, which is stored at –80°C.)