A28 Restriction Analysis

To demonstrate the existence of particular restriction sites in a DNA sample.

Prior Work

A sample of DNA must be available to characterize.

Background

• A DNA restriction endonuclease is an enzyme that recognizes a particular sequence of DNA bases, and catalyzes the cleavage of the double-stranded DNA. The cuts in each strand may be opposite one another, in which case the ends are said to be "blunt." More often, the cut in one strand is slightly displaced from that in another, leaving "sticky" ends which can experience complementary base pair recognition binding. A restriction site is a site where cleavage by a DNA restriction endonuclease takes place. Vectors (such as plasmids) may be cut by restriction enzymes. The site can be reconnected or a piece of new DNA may be inserted.
• In restriction analysis, recombinant DNA is treated with restriction enzymes to verify that a piece of DNA has been successfully inserted. A restriction map gives an indication of the relative locations of restriction sites in a DNA molecule.

Procedure

Ethidium bromide is very dangerous. Use this substance with great caution.

1. Prepare a gel electrophoresis tank. Tape the ends of a gel tray. Level the tray. Use a large level that does not touch the bed of the tray. A piece of aluminum foil permits leveling adjustment. Insert the comb into one of the sets of slots provided.
2. Add a preweighed mass of agarose to buffer. (Both buffer selection and gel concentration may vary.) Swirl the mixture. Cover. Heat in a microwave to boiling. Continue heating until clarified. Remove the hot container using a suitable protective device. Allow to cool to 50-60°C.
3. Add an aliquot of ethidium bromide. (Ethidium bromide is very toxic. Ethidium bromide (2,7-diamino-10-ethyl-9-phenyl-phenanthridinium bromide), a mutagen, has a planar molecular structure. It intercalates between the stacked bases of DNA. Radiation at 300 nm and 360 nm absorbed by the bound dye is emitted at 590 nm (reddish-orange).) Dispose of the pipet tip in a container reserved for ethidium bromide wastes. Treat the contents of this vessel with chlorine bleach to destroy the ethidium bromide prior to disposal. Pour the gel solution into the tray. Wait for the gel to harden (about 30 minutes).
4. Restrict the DNA sample while the gel is hardening. Add an aliquot of the sample to be restricted to a conical microcentrifuge tube. Add the necessary buffers and restriction enzymes. These materials often come in kits. The restriction enzymes are purchased in glycerol solution. Store the restriction enzyme in the freezer until the moment of use. Transfer the enzyme container to ice. Add a suitable aliquot of enzyme to the mixture. Because glycerol inhibits the restriction reaction, the total aliquot of enzyme is kept to less than 10% of the total reaction volume. A period of incubation will be required. Follow the protocol for the particular restriction enzyme being used.
5. Level the tank. Remove the comb very carefully; remove the tape. Place the tray in the tank. Fill the tank with 1x TPE buffer.
6. Add a dye marker such as bromphenol blue to the restricted sample. This usually contains glycerol to increase the density of the solution and thereby facilitate loading of the wells. Load samples into wells on the gel.
7. Cover the tank. Connect the power cords to the tank. Connect the power cords to a suitable power supply. Adjust to a suitable power setting. Apply power for the desired length of time. (This is usually until the leading edge, indicated by the migration of the marker dye, moves to the end of the gel.) Turn off the power. Disconnect the power cords, first from the power supply, and then from the tank.
8. Remove the tank cover. Remove the gel tray. Transfer the gel tray to a holding tray.
9. Wear a face shield to limit expsoure to ultraviolet radiation. Place the gel on the transilluminator. Examine the gel for bands. The bands are colorless. Ultraviolet light makes the bands appear as a pinkish fluorescence.
10. (A sample frame is displayed in the movie. The four pink fluorescing tracks that appear 20% up from the bottom of the screen and slightly to the right of center are the fluorescing bands.) An instant photograph may be taken of the fluorescing gel. Protocols for this photography depend upon the equipment and film.

Going Further

The restricted DNA is next assayed by Southern transfer (A-30) or quantitation. Electroelution (A-29) may be performed.

References