Molecular Biology. A Practical Guide - Velikov V.A. 2013

DNA Restriction
Restriction of Plasmid and Phage DNA

Enzyme Activity and Quantity. Each enzyme preparation has a specific activity. One unit of restriction enzyme activity completely hydrolyzes 1 µg of λ phage DNA in 1 h at 37°C. Commercial preparations typically have an activity of 10-20 units/µL and require dilution. DNA Hydrolysis is carried out using a 2-3-fold molar excess of the enzyme, i.e., 2-3 units are taken per 1 µg of DNA. Restriction Enzymes are stored at -20°C in 50% glycerol.

Buffers. Mg²⁺ ions are an absolutely essential cofactor for restriction enzyme activity. Buffer capacity at the required pH level is provided by Tris-HCl. β-Mercaptoethanol or dithiothreitol stabilizes the enzyme. High-salt (100 mM NaCl), medium-salt (50 mM NaCl), and low-salt (no NaCl) buffers are distinguished. Suppliers provide the required buffer in concentrated form along with the enzyme.

Reaction Mixture Volume. It is convenient to perform the restriction digest in a volume of 20-50 µL, where the DNA solution, 10× buffer, and enzyme are mixed in a microcentrifuge tube and brought to the final volume with bidistilled or deionized Water. Highly purified water is always used for enzymatic reactions.

Incubation Temperature and Time. Most enzymes operate at 37°C (except for those from thermophilic Bacteria). At room temperature, the reaction rate drops significantly. Incubation longer than 1 h may be necessary if Digestion has been incomplete for any reason. Incomplete restriction is indicated on the electrophoregram by DNA bands corresponding to those of the uncut control.

Materials and Equipment

pBR322 plasmid DNA solution (1 mg/mL), λ phage DNA solution (1 mg/mL), EcoRI restriction enzyme (10 units/µL), thermostat, Electrophoresis equipment.

Solutions

- 10× EcoRI restriction buffer: 500 mM Tris-HCl, pH 7.5; 100 mM MgCl₂; 1000 mM NaCl; 10 mM DTT.

- 10× gel loading buffer (Topic 3).

Procedure

1. Before Setting up the reaction (steps 6-12), you can use the RestrictionMapper program to find the number of EcoRI restriction sites in the λ phage DNA molecule and their sizes. This helps you anticipate what you will see on the electrophoregram. The nucleotide sequence of the phage can be retrieved from the GenBank database as described below (steps 2-5).

2. Open the homepage of the National Center for Biotechnology Information, US National Library of Medicine (http://www.ncbi.nlm.nih.gov/pubmed or http://www.pubmed.com). Enter the query "Enterobacteria phage lambda, complete genome". Select the "Nucleotide" tab and press "Enter". Copy the complete nucleotide sequence of bacteriophage λ, consisting of 48,502 bp (GenBank, NC 001416.1).

3. Open the RestrictionMapper program (http://www.restrictionmapper.org). Enter the phage λ nucleotide sequence into the designated box.

4. Select the "Selected individual Enzymes" option, choose the "EcoRI" enzyme, and click "Map Sites". You will see that there are 5 restriction sites, with the nucleotide position specified at each Cleavage point. There will be 6 fragments because the DNA is linear. You can find the sizes of all restriction fragments by clicking the "Virtual Digest" button. Record these sizes.

5. Repeat the procedure for the pBR322 plasmid by entering the query "Cloning vector pBR322", or refer to Appendices 3 and 4.

6. Prepare a 20 µL reaction mixture in a 1.7 mL plastic tube: 10× buffer – 2 µL, DNA solution (1 mg/mL) – 1 µL, water – 16.7 µL.

7. Mix the contents thoroughly by pipetting or vortexing, and spin down droplets from the sides of the tube with a brief centrifugation.

8. Remove the enzyme from the freezer and immediately place the tube on ice. Using a micropipette, draw the required volume of the protein solution containing 3 activity units (0.3 µL). Add the enzyme to the DNA sample and immediately return the restriction enzyme to the -20°C freezer. Mix and collect the liquid from the walls of the tube by a brief centrifugation.

9. Incubate the sample for 1 h ^a at the recommended temperature of 37°C.

10. Stop the reaction by adding 1 µL of 200 mM EDTA or by heating at 65°C for 5-10 min ^b.

11. Add 1/10 volume of 10× gel loading buffer to the digested DNA preparation, mix by pipetting, and load the DNA into the gel wells. In this buffer, the resulting DNA samples can be stored in a refrigerator for some time.

12. Perform DNA electrophoresis at 100 V for 1.5-2 h and evaluate the results by visualizing the gel under UV light ^c. Photograph the gel.

Notes

^a The incubation time may be extended to 2 hours or more to ensure complete digestion. Commercial enzyme preparations typically do not contain nuclease contaminants and thus lack nonspecific nuclease activity. However, excessively long incubation with certain restriction enzymes may lead to star activity, i.e., a decrease in DNA digestion Specificity (for EcoRI* — NAATTN). This will result in a "smear" instead of distinct bands in the gel.

^b In addition, heat-treated preparations provide better Separation of restriction fragments with close molecular weights, which are sometimes prone to "sticking". Often, such DNA fragments only begin to separate into two distinct bands near the bottom of the gel. This can be observed, for example, with the 5,804 and 5,643 bp EcoRI fragments of phage λ DNA.

^в The number of restriction sites in circular DNA corresponds precisely to the number of restriction fragments generated. Complete EcoRI digestion of the plasmid pBR322 yields a single restriction fragment. Digestion of linear phage λ DNA (containing 5 sites) yields 6 restriction fragments with lengths of 21226, 7421, 5804, 5643, 4878, and 3530 bp. If a greater number of fragments is visible in the sample (pattern), it invariably indicates incomplete DNA digestion, i.e., the presence of intermediate digestion products (“partial digestion”).