RESTRICTION ENDONUCLEASES SECTION I Digestion of DNA with Restriction UNIT 3.1 Endonucleases Restriction endonucleases recognize short DNA sequences and cleave double-stranded DNA at specific sites within or adjacent to the recognition sequences. Restriction endonuclease cleavage of DNA into discrete fragments is one of the most basic procedures in molecular biology. The first method presented in this unit is the cleavage of a single DNA sample with a single restriction endonuclease (see Basic Protocol). A number of common applications of this technique are also described. These include digesting a given DNA sample with more than one endonuclease (see Alternate Protocol 1), digesting multiple DNA samples with the same endonuclease (see Alternate Protocol 2), and partially digesting DNA such that cleavage only occurs at a subset of the restriction sites (see Alternate Protocol 3). A protocol for methylating specific DNA sequences and protecting them from restriction endonuclease cleavage is also presented (see Support Protocol). A collection of tables describing restriction endonucleases and their properties (including information about recognition sequences, types of termini produced, buffer conditions, and conditions for thermal inactivation) is given at the end of this unit (see Table 3.1.1, Table 3.1.2, Table 3.1.3, and Table 3.1.4). DIGESTING A SINGLE DNA SAMPLE WITH A SINGLE RESTRICTION BASIC ENDONUCLEASE PROTOCOL Restriction endonuclease cleavage is accomplished simply by incubating the enzyme(s) with the DNA in appropriate reaction conditions. The amounts of enzyme and DNA, the buffer and ionic concentrations, and the temperature and duration of the reaction will vary depending upon the specific application. Materials DNA sample in H 2O or TE buffer ( APPENDIX 2 ) 10 × restriction endonuclease buffers (see recipe) Restriction endonucleases (Table 3.1.1 and Table 3.1.3) 10 × loading buffer ( UNIT 2.5 ) 0.5 M EDTA, pH 8.0 (optional; APPENDIX 2 ) Additional reagents and equipment for agarose or polyacrylamide gel electrophoresis ( UNIT 2.5 or UNIT 2.7 ), DNA extraction (optional; UNIT 2.1 ), and ethanol precipitation (optional; UNIT 2.1 ) 1. Pipet the following into a clean microcentrifuge tube: x µl DNA (0.1 to 4 µg DNA in H 2O or TE buffer) 2 µl 10 × restriction buffer (Table 3.1.1) 18 − x µl H 2O. A 20- ìl reaction is convenient for analysis by electrophoresis in polyacrylamide or agarose gels. The amount of DNA to be cleaved and/or the reaction volume can be increased or decreased provided the proportions of the components remain constant. 2. Add restriction endonuclease (1 to 5 U/ µg DNA) and incubate the reaction mixture 1 hr at the recommended temperature (in general, 37°C). Enzymatic Manipulation of DNA and RNA Contributed by Kenneth D. Bloch and Barbara Grossmann 3.1.1 Current Protocols in Molecular Biology (1995) 3.1.1-31.21 Copyright © 2000 by John Wiley & Sons, Inc. Supplement 31 In principle, 1 U restriction endonuclease completely digests 1 ìg of purified DNA in 60 min using the recommended assay conditions. However, crude DNA preparations, such as those made by rapid procedures ( UNIT 1.6 ), often require more enzyme and/or more time for complete digestion (see Critical Parameters). The volume of restriction endonuclease 1 added should be less than ⁄10 the volume of the final reaction mixture, because glycerol in the enzyme storage buffer may interfere with the reaction. 3. Stop the reaction and prepare it for agarose or acrylamide gel electrophoresis ( UNIT 2.5 or UNIT 2.7 ) by adding 5 µl (20% of reaction vol) 10 × loading buffer. The reaction can also be stopped by chelating Mg 2+ with 0.5 ìl of 0.5 M EDTA (12.5 mM final concentration). If the digested DNA is to be used in subsequent enzymatic reactions (e.g., ligation or “filling-in” reactions), addition of EDTA should be avoided. Alternatively, many enzymes can be irreversibly inactivated by incubating 10 min at 65 °C (see Table 3.1.1). Some enzymes that are partially or completely resistant to heat inactivation at 65 °C may be inactivated by incubating 15 min at 75 °C. When the enzyme(s) is completely resistant to heat inactivation, DNA may be purified from the reaction mixture by extraction with phenol and precipitation in ethanol ( UNIT 2.1 ). Alternatively, DNA may be purified conveniently using a silica matrix suspension as described in UNIT 2.1 (also commercially available as Geneclean from Bio101.) ALTERNATE DIGESTING DNA WITH MULTIPLE RESTRICTION PROTOCOL 1 ENDONUCLEASES It is often desirable to cleave a given DNA sample with more than one endonuclease. Two or more enzymes may be added to the same reaction mixture if all are relatively active in the same buffer and at the same temperature. Many enzymes are active in a wide variety of buffer solutions. It is frequently possible to choose a standard buffer solution in which two or more enzymes will retain activity (see Commentary and Table 3.1.2). Alternatively, most restriction endonucleases and some DNA-modifying enzymes are active to some extent in potassium glutamate– and potassium acetate–based buffers (see recipe for 10 × restriction endonuclease buffers). Hence, these buffers may be useful for digesting DNA with multiple enzymes. However, if the reaction conditions needed are too dissimilar, follow the procedure below. 1. Digest the DNA with the enzyme(s) that is active at the lower NaCl concentration (see Basic Protocol, steps 1 and 2). If optimal digestion conditions differ only in incubation temperature, cleave the DNA with one enzyme, then shift the temperature and add the second enzyme (the order of cleavage does not matter; Table 3.1.1). However, many enzymes with optimal activity at high temperatures are also active at 37 °C. In these cases, the enzymes can be added simultane- ously and the reaction mixture incubated at 37 °C (it may be necessary to add more of the “high-temperature” enzyme than usual). 2. For enzymes active at higher salt concentrations, add 1 M NaCl (1 to 3 µl for a 20- µl reaction) so that the final concentration is suitable for digestion by the next enzyme(s). Add enzyme(s) for the second reaction and incubate appropriately. Purification of the DNA fragments between digestions is the most reliable method to ensure complete, multiple digestions. However, it is much more laborious and is rarely necessary. 3. Stop the reaction for electrophoretic analysis or further enzymatic treatment (see Basic Protocol, step 3). Digestion of DNA with Restriction Endonucleases 3.1.2 Supplement 31 Current Protocols in Molecular Biology DIGESTING MULTIPLE SAMPLES OF DNA ALTERNATE PROTOCOL 2 This procedure minimizes the number of pipetting steps when multiple samples are to be digested with the same enzyme(s) and, hence, saves time. More importantly, by minimiz- ing the number of transfers from the tube containing the restriction enzyme, the potential for contamination of the enzyme is reduced. 1. For each sample to be tested, add a constant volume of DNA to a separate microcen- trifuge tube. It is critical to use a different pipet tip for each DNA sample in order to prevent cross-contamination. 2. Prepare a “premix solution” containing sufficient 10 × restriction endonuclease buffer and water for digesting all the samples. Place solution on ice. For example, if ten 3- ìl samples of DNA are each to be digested in a 20- ìl reaction mixture, the premix will contain 20 ìl of 10 × restriction buffer and 150 ìl water. It is prudent to make up enough solution for at least one more sample than is to be tested. 3. Add sufficient restriction endonuclease(s) for digesting all the samples. Mix quickly by flicking the tube and replace on ice. The solution to which the enzyme is added should not be more concentrated than 3 × buffer. 4. Add the appropriate amount of solution containing the restriction endonuclease (17 µl for above example) to each tube of DNA and incubate the reactions 1 hr at the appropriate temperature. For most analytical purposes, the same pipet tip can be used to dispense the restriction endonuclease solution provided that care is taken to avoid direct contact with the DNA at the bottom of the tubes. For preparative purposes, it is advisable to use a different pipet tip for each sample. 5. Stop the reactions for electrophoretic analysis or further enzyme digestion (see Basic Protocol, step 3). PARTIAL DIGESTION OF DNA WITH RESTRICTION ALTERNATE ENDONUCLEASES PROTOCOL 3 For some purposes, it is useful to produce DNA that has been cleaved at only a subset of the restriction sites. This is particularly important for cloning segments of DNA in which the site(s) used for cloning is also present internally within the segment, and is also useful for restriction mapping ( UNIT 3.3 ). Partial cleavage is accomplished using reduced (and therefore limiting) concentrations of restriction enzyme. Here a set of serial dilutions of enzyme is set up so that one or more of the conditions used is likely to produce the appropriate partial digestion product. If necessary, the procedure can be used for analytical purposes, and the reaction can be repeated and/or scaled up using digestion conditions that have been empirically determined. Partial cleavage can also be achieved by varying the time of digestion, but this is more tedious and less reliable. 1. Make up a 100- µl reaction mixture containing DNA in 1 × restriction enzyme buffer. 2. Divide up reaction mixture such that tube 1 contains 30 µl, tubes 2 to 4 contain 20 µl, and tube 5 contains 10 µl. Place tubes on ice. 3. Add the selected restriction endonuclease (3 to 10 U/ µg DNA) to tube 1, mix quickly by flicking the tube, and place the tube back on ice.