Restriction Enzyme Digest and Gel Electrophoresis Field Trip

Background Information

Restriction enzymes are proteins that cut double stranded DNA at specific recognition sites. They were first proposed in the early 1960s by W. Arber and D. Dussoix to explain the way bacteriophages (viruses that infect bacteria) could infect some strains of bacteria but not others. Restriction enzymes are isolated from bacteria. The bacteria use them as protection against the invasion of foreign DNA. In 1968, W. Arber and S. Linn, and then M. Meselson and R. Yuan, purified similar enzymes that were able to cut DNA, but these early enzymes cleaved the DNA at random positions. In 1970, H.O. Smith, K. W. Wilcox and T. J. Kelly purified and characterized the recognition and cleavage site of a more useful enzyme, Hind II, an enzyme that cut at a specific recognition site every time. This enzyme was used by Daniel Nathans to cut the circular genome of Simian Virus 40 (SV40) to generate the first restriction map. The ability to cut DNA at specific sequences became the first step toward molecular cloning. For their groundbreaking discoveries, Arber, Smith and Nathans were awarded the Nobel Prize in Physiology or Medicine in 1978.

In the Restriction Enzyme Digest and Gel Electrophoresis of DNA field trip, the students cut lambda DNA with restriction enzymes and use gel electrophoresis to visualize the DNA. Molecular biology laboratory skills and equipment, as well as laboratory safety, will be discussed and used in this lab.

Lambda DNA is used in this experiment. Lambda is a bacteriophage. The isolated DNA is linear, 48,502 base pairs long, and has recognition sites for many different restriction enzymes. It is commonly used for molecular weight, size markers in gel analysis of DNA, and as a substrate in restriction enzyme activity assays.

In this lab, we cut lambda DNA with different restriction enzymes and enzyme blends. These reactions are known as restriction digests. Restriction enzymes act like scissors to cut DNA into pieces. Different restriction enzymes (and there are hundreds) recognize and cut different DNA sequences. When the DNA is cut, the sizes of the DNA fragments correspond to the distances (in base pairs) between restriction sites. The reaction is incubated at the enzyme's optimum temperature to digest the DNA. This usually takes about 45 minutes. During this time agarose gels will be prepared.

Purified agarose is a powder that is insoluble in water (or buffer) at room temperature but dissolves in boiling water (or buffer). As it cools, agarose undergoes polymerization. The sugar polymers crosslink with each other and cause the solution to "gel", much like JELLO. Higher concentrations of agarose give firmer gels. If you were inside a gel, it would resemble a very dense spider web. If you were a small fragment of DNA, then you could easily crawl through the spaces between the webs (they are too tough for you to just pull out of the way) but as you increase in length, it gets harder and harder for you to fit through the spaces. We use a 0.8% agarose gel in 1x sodium borate buffer for this experiment. The agarose is kept molten at 55° C until the gels are poured. Gels are prepared by pouring the molten agarose into casting trays around a six-well comb and allowing them to solidify. The comb is removed from the solidified gel and the DNA is loaded into the wells left by the comb.

The gel box is prepared by pouring 1x sodium borate buffer into the chambers and placing the gel in the box. The digested DNA and some DNA markers will be mixed with a multi-purpose-dye called FOTO/Vision ^TM. FOTO/Vision^TM serves as a loading and tracking dye during electrophoresis. It is also a safe-stain, replacing ethidium bromide as the DNA-binding dye used for visualization of DNA bands in the resulting gel. The samples are loaded into the agarose gel wells and then electrophoresed by connecting the gel box to a power supply.

Electrophoresis is a laboratory technique used the to separate charged molecules. DNA is negatively charged and moves, under the force of an electric current, through the matrix of the agarose. Molecules separate by size, with the smaller ones moving more rapidly through the gel than the large ones.

After running the gel, the DNA is visualized using medium wavelength ultraviolet (UV) light (we always wear safety glasses while using a UV light source). A component of FOTO/Vision^TM binds to the DNA during electrophoresis. When the gel is exposed to UV light, the DNA bands fluoresce in a distinctive pattern of DNA fragments, one band for each different size fragment. We take a photograph of the gel so that it is easier to study the banding pattern.

If you have any questions or would like more information before you bring your students to the BTC Institute for this field trip, please give us a call. Alternatively, bring your questions along and we can discuss them during the lab. We look forward to seeing you and your group on your scheduled field trip day. Thank you for your interest in the BTC Institute's Biotechnology Field Trips program!