Restriction Endonuclease Digestion of DNA. Duncan R. Smith. 1. Introduction. The ability to cleave DNA at specific sites is one of the cornerstones of today's.
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Restriction Endonuclease Digestion of DNA Duncan R. Smith
1. Introduction The ability to cleave DNA at specific sites is one of the cornerstones of today's methods of DNA manipulation. Restriction endonucleases are bacterial enzymes that cleave duplex DNA at specific target sequences with the production of defined fragments. These enzymes can be purchased from the many manufacturers of biotechnology products. The nomenclature of enzymes is based on a simple system, proposed by Smith and Nathans (1). The name of the enzyme (such as BamHl, EeaRl, and so on) tells us about the origin of the enzyme but does not give us any information about the specificity of cleavage (see Note 1). This has to be determined for each individual enzyme. The recognition site for most of the commonly used enzymes is a short palindromic sequence, usually either 4, 5, or 6 bp in length, such as AGCT (for AZul), GAATTC (for EeaRl), and so on. Each enzyme cuts the palindrome at a particular site, and two different enzymes may have the same recognition sequence but cleave the DNA at different points within that sequence. The cleavage sites fall into three different categories, either flush (or blunt) in which the recognition site is cut in the middle, or with either 5'- or 3'-overhangs, in which case unpaired bases will be produced on both ends of the fragment. For a comprehensive review of restriction endonucleases, see Fuchs and Blakesley (2). 2. Materials 1. lOX stock of the appropriate restriction enzyme buffer (see Note 2). 2. DNA to be digested (see Notes 3 and 4) in either water or TE (10 mM Tris-HCl pH 8.3, 1 mM ethylenediaminetetraacetic acid [EDTA]). 3. Bovine serum albumin (BSA) at a concentration of 1 mg/mL (see Note 5). 4. Sterile distilled water (see Note 6). 5. The correct enzyme for the digest (see Note 7). 6. 5X Loading buffer: 50% (v/v) glycerol, 100 mM Na2EDTA, pH 8, 0.125% (w/v) bromophenol blue, 0.125% (w/v) xylene cyanol. 7. 100 mM Spermidine (see Note 8).
3. Methods 1. Thaw all solutions, with the exception of the enzyme, and then place on ice. From: The Nucleic Acid Protocols Handbook Edited by: R. Rapley © Humana Press Inc., Totowa, NJ
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2. Decide on a final volume for the digest, usually between 10 and 50 ~ (see Note 9), and then into a sterile Eppendorftube, add 1110 vol ofreaction buffer, 1110 vol BSA, between 0.5 and 1 !Jg of the DNA to be digested (see Note 3), and sterile distilled water to the final volume. 3. Take the restriction enzyme stock directly from the -20 c C freezer, and remove the desired units of enzyme (see Notes 7 and 10) with a clean sterile pipet tip. Immediately add the enzyme to the reaction and mix (see Note 11). 4. Incubate the tube at the correct temperature (see Note 12) for approx 1 h. Genomic DNA can be digested overnight. 5. An aliquot of the reaction (usually 1-2~) may be mixed with a 5X concentrated loading buffer and analyzed by gel electrophoresis (see Chapter 13).
4. Notes I. Enzymes are named according to the system proposed by Smith and Nathans (1), in which enzymes are named according to the bacteria from which they are first purified. Therefore, for example, a restriction enzyme purified from Providencia stuartii, would be identified by the first letter of the genus name (in this case, Providencia and hence P) and the first two letters of the specific epithet (in this case, stuartii and hence st) joined together to form a three-letter abbreviation: Pst. The first restriction enzyme isolated from this source of bacteria would therefore be called PstI (with the number in Roman numerals), and the second PstII, and so on. Note, however, that the name of the enzyme gives no information about the specificity of cleavage, which must be determined from one of the numerous lists of enzymes and cleavage specificities (the catalog of most suppliers of restriction enzymes will provide extensive information about restriction enzymes, such as specificity of cleavage, optimal reaction conditions, number of cleavage sites in common DNA templates, and so on, and these catalogs should be treated as valuable sources of information). 2. Each enzyme has an optimal reaction buffer. The recommended reaction conditions are normally to be found on the manufacturer's assay sheet. In practice, many enzymes share common conditions, and it is possible to make up reaction buffers that are suitable for a number of enzymes. The vast majority of enzymes will work in one of three buffers, either a high-, 10w-, or medium-salt buffer, recipes for which are given as follows. These buffers are normally made as a lOX stock and then 1110 final volume is added to each digest. Great care must be taken in matching the buffer to the enzyme, since the wrong buffer can give either a dramatically reduced activity, altered specificity, or no activity at all. Several manufacturers of restriction enzymes now provide the correct buffer with their enzymes as an added benefit, and it is recommended that where these buffers are provided, they should be used. a. High-salt buffer (IX): 100 mM NaCI, 50 mM Tris-HCI, pH 7.5, 10 mM MgCI 2 , 1 mM dithiothreitol (DTT). b. Medium-salt buffer (IX): 50 mM NaCI, 10 mM Tris-HCI, pH 7.5,10 mM MgCI 2 , 1 mMDTT.
c. Low-salt buffer (IX): 10 roM Tris-HCI, pH 7.5, 10 roM MgCI 2 , 1 roM DTT. In addition, two "universal buffers" are occasionally used, which are buffers in which all restriction enzymes have activity, although in some cases, activity can be reduced to only 20% of optimal activity. These are the potassium-glutamate (3) and potassiumacetate (4) buffers. These buffers can be particularly useful when a piece of DNA must be digested by two enzymes having very different optimal buffers. 3. The amount of DNA to be digested depends on subsequent steps. A reasonable amount for a plasmid digestion to confirm the presence of an insertion would be 500 ng-l Ilg,
Restriction Endonuclease Digestion of DNA
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depending on the size of the insert. The smaller the insert, the more DNA should be digested to enable visualization of the insert after agarose gel analysis. The DNA to be digested should be relatively pure and free from reagents, such as phenol, chloroform, alcohols, salts, detergents, and chelating agents. Any trace amounts of these chemicals will inhibit or inactivate the restriction endonuclease. BSA is routinely included in restriction digests to stabilize low protein concentrations and to protect against factors that cause denaturation. Good-quality sterile distilled water should be used in restriction digests. Water should be free of ions and organic compounds, and must be detergent free. An enzyme unit is defined as the amount of enzyme required to digest 1 f.lg of a standard DNA in 1 h under optimal temperature and buffer conditions. The standard DNA used is normally 'A DNA. Hence, for EcoRI, e.g., there are five sites for this enzyme in 'A. If one is digesting PBR322, which has one site, with 1 U of enzyme for 1 h, this is actually a fivefold overdigestion. Digests of genomic DNA are dramatically improved by the inclusion of spermidine in the digest mixture to a final concentration of 1 mM since the polycationic spermidine binds negatively charged contaminants. Note that spermidine can cause precipitation of DNA at low temperatures, so it should not be added while the reaction is kept on ice. The smallest practical volume in which to undertake a restriction digest is 10 IlL. Below this, pipeting errors can introduce significant errors in the reaction conditions. This volume also allows the entire digest to be loaded onto a small agarose gel after the addition of the stop/loading buffer. If the stock DNA concentration is too dilute to give 0.5-1 f.lg in 5-6 IlL, then the reaction can be scaled up to 20-50 IlL. If double digestion is to be undertaken (i.e., digestion with two different enzymes), then 20 IlL is the recommended minimum volume, 1 IlL of each enzyme can be added, and the glycerol concentration is kept low (see Note 10). Many enzymes are susceptible to the presence of glycerol. The majority of stock enzymes are provided in approx 50% (v/v) glycerol. A reaction digest in which more than approx 10% (v/v) glycerol is present can give cleavage at different sites from the normal (the so-called star activity). For this reason, it is advisable to keep the enzyme total reaction volume ratio at I: 10 or lower. Similar star activity can result from incorrect salt concentrations. Stock restriction enzymes are very heat labile and so should be removed from -20 o e storage for as short a time as possible and placed on ice. Note that the incubation temperature for the vast majority of restriction endonucleases is 37°e but that this is not true for all enzymes. Other enzymes, such as TaqI and SmaI, require different optimal temperatures (in this case 65 and 25°e, respectively). It is wise, therefore, to check new or unfamiliar enzymes before use. If large-scale preparative digests are to be undertaken (l00-500 IlL reaction mixes), then the reaction is scaled up accordingly. However, care must be taken to ensure that the reaction components are fully mixed, especially with regard to the viscous constituents, such as DNA solutions and stock restriction enzymes. For all volume digests, vortexing should be avoided, as this can significantly reduce the activity of the enzyme. For small volumes, mixing can be achieved by tapping or gently flicking the tube with a finger (often followed by a brief 1-5 s spin in an Eppendorf centrifuge to deposit the reaction at the bottom of the tube). For larger volumes, mixing can be achieved by gentle pipeting, taking liquid from the bottom of the reaction volume, and mixing at the top of the reaction volume until a homogenous solution is obtained.
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References 1. Smith, H. O. and Nathans, D. (1973) A suggested nomenclature for bacterial host modification and restriction systems and their enzymes. J. Mol. BioI. 81,419--423. 2. Fuchs, R. and Blakesley, R. (1983) Guide to the use of Type 11 restriction endonucleases. Methods Enzymol. 100, 3-38. 3. McClelland, M., Hanish, J., Nelson, M., and Patel, Y. (1988) KGB: a single buffer for all restriction endonucleases. Nucleic Acids Res. 16, 364. 4. O'Farrell, P. H., Kutter, E., and Nakanishe, M. (1980) A restriction map of the bacteriophage T4 genome. Mol. Gen. Genet. 170,411--435.
