Restriction Enzymes Resource

2.3 Restriction Enzyme Substrate Considerations

1. Substrate Source and Structure
2. Substrate Quality
3. Recognition Site Density
4. References

A. Substrate Source and Structure

Substrates commonly used for restriction enzyme digestion include phage DNA, plasmid DNA, genomic DNA, PCR^(a) products and double-stranded oligonucleotides. 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 0.02-0.2µg/µl. Substrate structural variations, concentration and special considerations are discussed below according to DNA type.

Lambda DNA: Lambda DNA is a linear DNA that is an industry standard for the measurement and expression of unit activity for most restriction enzymes. In general, one unit is sufficient to cut 1µg of lambda DNA in 1 hour under optimal reaction conditions in a reaction volume of 50µl. In lambda DNA, the cos ends, (12-base, complimentary, single-stranded overhangs at the end of each molecule) may re-anneal during digestion. This can give the appearance that digestion is incomplete. To avoid this problem, heat the DNA at 65°C for 5 minutes prior to electrophoresis to melt ends that have annealed.

Plasmid DNA: Circular, supercoiled plasmid DNA typically ranges from 3-10kb in size. 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.

Genomic DNA: Digestion of genomic DNA can be difficult due to methylation and viscosity. If methylation is a concern, consider using isochizomers with different methylation sensitivities (see Methylation Sensitivity of Isoshizomer/Neoschizomer Pairs). Viscosity can be adjusted by increasing the reaction volume. Genomic DNA often digests more efficiently when it is diluted to a minimum concentration of 10µg per 50-200µl. If this is not possible, heating the DNA at 65^ºC for ten minutes prior to the addition of the restriction enzyme can enhance activity (3). Addition of spermidine to final concentration of 1-5mM 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 0.1mg/ml may also improve enzyme activity.

PCR Products: PCR-amplified DNA may be digested with restriction enzymes that have recognition sequences within the amplified sequence or in the primer regions. 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. Enzymes with low overdigestion values (<12 units/16 hours) should be avoided in overnight digestions, as star activity or trace contaminants present in these enzymes may lead to problems. 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. Restriction enzymes require varying amounts of flanking DNA around the recognition site, usually 1-3 bases but occasionally more (See Digestion of Sites Close to the End of Linear DNA). 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. Use of proofreading enzymes in PCR may also complicate the situation as these enzymes are capable of degrading the 3´ ends of amplimers, interfering with complete digestion by restriction enzymes. The use of high dNTP concentrations and immediate cooling to 4°C after PCR will reduce such degradation. 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. This problem can be easily avoided by purifying the PCR fragment prior to restriction enzyme digestion using the Wizard^® PCR Preps DNA Purification System^(b) (Cat.# A7170).

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. Again, longer digestion times and/or more enzyme may be needed. Enzymes with a low overdigestion specification (12 units/16 hours) should be avoided in overnight digestions.

Single-Stranded DNA: Cleavage of single-stranded DNA, although at a greatly reduced rate compared with double-stranded DNA, has been reported for a few restriction enzymes (6). 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.g., M13, f1, single-stranded phiX174) (7,8). Therefore, these enzymes are not digesting single-stranded DNA, rather individual sites that are in the duplex form.

DNA-RNA Hybrids: Digestion of DNA-RNA hybrid molecules has been described for several restriction enzymes (Alu I, EcoR I, Hae III, Hha I, Hind III, Msp I, Sal I, Tha I) (9). In these cases, the DNA strand of the hybrid was digested in the identical place as duplex DNA. Digestion required 20 to 50-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 10-fold. A small number of enzymes (e.g., Nae I, Hpa II, Sac II, Nar I, EcoR II) exhibit more pronounced site preferences and are designated Type IIe. See Site Preferences and Turbo™ Restriction Enzymes for further information.

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 (10). Often, isoschizomers differ in their methylation sensitivity. Refer to Table 3.6 for further information.

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B. Substrate Quality

General Quality of the DNA Substrate and Effect on Digestion: Highly purified DNA is required for efficient restriction enzyme digestions. Contaminants commonly used during the purification of DNA such as protein, phenol, chloroform, ethanol, EDTA, SDS, CsCl and NaCl may interfere with restriction enzyme performance if not eliminated prior to digestion (1). Organic solvents may denature the enzyme and additional salt contamination may decrease enzyme activity. Because contaminants are usually dose dependent, inhibitory effects will increase with the volume of DNA added to the digestion reaction. Protein contaminants in DNA can include nucleases that are activated by the addition of Mg²⁺ or salt in the restriction enzyme buffer. The presence of such nuclease activity will result in degradation of the substrate DNA, evidenced upon electrophoresis as a smear or a loss of DNA compared with a control sample of untreated DNA. Other potential contaminants include DNA binding proteins, which may sterically interfere with the ability of the enzyme to efficiently find its recognition site and/or retard electrophoretic mobility of the restriction fragments.

DNA samples are often stored in Tris-EDTA (TE) buffer since EDTA inactivates most nucleases that co-purify with DNA by chelating the divalent cations required for their activity. However, restriction enzymes require divalent cations for activity and may be inhibited if too much EDTA is present in the final reaction. Low concentrations of EDTA (less than 0.05mM) introduced into the restriction enzyme reaction as in the DNA storage buffer do not substantially affect restriction enzyme activity.

Some of the most commonly encountered problems for specific DNA preparations are discussed below and suggestions for optimizing the performance of restriction enzymes on these substrates are provided.

Miniprep DNA: DNA purified from minipreps can be of poor quality due to contaminants such as phenol, chloroform, protein or RNA. In addition, some bacterial strains used to amplify plasmid DNA (e.g., HB101) contain a greater amount of nuclease than others (e.g., JM109). Such enzymatic contaminants may only become apparent when activated by the Mg²⁺ and salt present in restriction digest buffers. Phenol/chloroform extraction may be required to remove these contaminants even after CsCl purification. Dialysis and/or multiple ethanol precipitations with 2.5M ammonium acetate and drying can remove many of the interfering substances introduced during purification. Ammonium acetate, which is a volatile substance, has unique and beneficial properties compared with other salts used for nucleic acid precipitation, but must be used in substantially higher concentrations (1).

Alternatively, the Wizard^® Plus SV Minipreps DNA Purification Systems^(c) (Cat.# A1330) provide an easy and effective way to isolate and purify DNA, free of salt or macromolecular contaminants. The addition of spermidine to a final concentration of 1mM and/or BSA to a final concentration of 0.1mg/ml can also improve digestion of poor quality miniprep DNA.

Genomic DNA: Genomic DNA frequently contains more contaminants than plasmid DNA. Best results are obtained when the absorbance ratios at A₂₆₀/A₂₈₀ are at least 1.8. Spermidine can be added to a final concentration of 1mM and/or BSA to a final concentration of 0.1mg/ml to improve digestion of poor quality genomic DNA. For further information see Digestion of High Molecular Weight DNA.

Genomic DNA Embedded in Agarose plugs: Pulsed field gel electrophoresis permits the resolution of extremely large DNA fragments. 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 50-1000kb. 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 (11). Most restriction enzymes can cut DNA embedded in agarose provided that more enzyme and longer incubation times are used. A good rule of thumb is to use 5-10 units of enzyme per microgram of DNA and to avoid using restriction enzymes with low overdigestion values (<20 units/16 hours), which can cause problems during longer incubations with excess enzyme. For further information, refer to Digestion of High Molecular Weight DNA.

Genomic DNA Purified From Blood. 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. The absorbance ratios at A₂₆₀/A₂₈₀ should be at least 1.8, indicating that protein has been removed efficiently. A number of rapid DNA purification protocols have been written that do not require separation of white cells from red cells (12,13). These techniques can yield good quality DNA from small volumes of blood, but the DNA obtained after scale-up may be of poorer quality. For larger blood samples, a technique that separates white blood cells from red blood cells, such as pelleting red blood cells through a Ficoll^® gradient, is recommended prior to DNA purification.

Promega offers the Wizard^® Genomic DNA Purification Kit (Cat.# A1120) for the isolation of genomic DNA from white blood cells (with reagents/protocol for removal of red cells), tissue cultured cells, animal tissue, plant tissue and Gram-positive and Gram-negative bacteria. DNA purified with this system is suitable for digestion with restriction enzymes.

PCR Products: Contaminants in PCR such as salts, glycerol, and primer dimers can inhibit restriction enzyme activity. The Wizard^® PCR Preps DNA Purification System (Cat.# A7170) provides a reliable method for purification of double-stranded PCR-amplified DNA from any salts or macromolecular contaminants.

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C. Recognition Site Density

Restriction enzyme activity units are usually defined based on a one-hour digest of 1µg of lambda DNA. 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 EcoR I while keeping the substrate mass and incubation time constant.

Table 2.3. Differences in Substrate Recognition Sites for EcoR I.

*Based on 650 Daltons per base pair of DNA.
**Enzymes differ in their ability to digest supercoiled vs. linear substrates.

D. References

1. Fuchs, R. and Blakesley, R. (1983) Guide to the use of type II restriction endonucleases. Meth. Enzymol. 101, 3.
2. Wells, R., Klein, R. and Singleton, C.K. (1981) In The Enzymes XIV, 157.
3. Hinds, K., Shamblott, M. and Litman, G. (1991) In Methods in Nucleic Acid Research Karam, J., Chao, L. and Warr, G. eds., CRC Press.
4. Bloch, K. (1987) In Current Protocols in Molecular Biology, Ausubel, F.M. et al., eds., Green Publishing Associates.
5. Turbett, G.R. and Sellner, L.N. (1996) Digestion of PCR and RT-PCR products with restriction endonucleases without prior purification or precipitation. Promega Notes 60, 23.
6. Yoo, O.J. and Agarwal, K. L. (1980) Cleavage of single strand oligonucleotides and bacteriophage phiX174 DNA by Msp I endonuclease. J. Biol. Chem. 255, 10559
7. Nevendorf, S. and Wells, R. (1980) In Gene Amplification and Analysis: Restriction Endonucleases. Vol. I, Chiriklian, J., ed., Elsevier, North Holland.
8. Blakesley, R.W. et al. (1977) Duplex regions in "single-stranded" phiX174 DNA are cleaved by a restriction endonuclease from Haemophilus aegyptius. J. Biol. Chem. 252, 7300.
9. Molloy, P.L. and Symons, R.H. (1980) Cleavage of DNA.RNA hybrids by type II restriction enzymes Nucl. Acids Res. 8, 2939.
10. McClelland, M. et al. (1994) Effect of site-specific modification on restriction endonucleases and DNA modification methyltransferases. Nucl. Acids Res. 22,3640.
11. McClelland, M. et al. (1987) Restriction endonucleases for pulsed field mapping of bacterial genomes. Nucl. Acids Res. 15, 5985.
12. Miller, S.A., Dykes, D.D. and Polesky, H.F. (1988) A simple salting out procedure for extracting DNA from human nucleated cells. Nucl. Acids Res. 16, 1215.
13. Grimberg, J. et al. (1989) A simple and efficient non-organic procedure for the isolation of genomic DNA from blood. Nucl. Acids Res. 17, 8390.

^(a)The PCR process is covered by patents issued and applicable in certain countries. Promega does not encourage or support the unauthorized or unlicensed use of the PCR process.
^(b)Licensed under U.S. Pat. No. 5,075,430.
^(c)U.S. Pat. Nos. 5,658,548 and 5,808,041, and Australian Pat. No. 689815 have been issued to Promega Corporation for nucleic acid purification on silica gel and glass mixtures.

Turbo is a trademark of Promega Corporation. Wizard is a trademark of Promega Corporation and is registered with the U.S. Patent and Trademark Office.
Ficoll is a registered trademark of Amersham Pharmacia Biotech Ltd.

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