The sequence of a DNA molecule determines the distribution of recognition sites for REs. Hundreds of REs with unique specifities have been described, so researchers can use the distribution of these recognition sites in a DNA molecule to construct a “map” of the sequence. In these experiments, DNA samples are digested with various REs to produce a restriction map, a collection of smaller restriction fragments that have been cleaved at either end by the RE. The molecules in the digest are then separated by agarose gel electrophoresis (Chapter 8). From the sizes of the restriction fragments that are resolved on the gel, investigators are able to identify the original DNA molecule used in the restriction digest.
Careful planning is required for meaningful restriction maps. The first step in a mapping experiment is to identify the sizes of restriction fragments that will be generated from a target DNA molecule with different REs. A variety of software programs generate these restriction maps and provide tabular data with details about the lengths and positions of the restriction fragments in the DNA sequence. The list of enzymes that cut a particular sequence is always impressive,
but only a few enzymes usually turn out to be practical for the purpose of the experiment. When choosing REs for a restriction map, there are many things to consider:
- How many restriction fragments will be generated?
- What are the predicted sizes of the restriction fragments?
- Will all the restriction fragments be clearly resolved on 1% agarose gels?
- Will the RE generate a distinctive set of fragments from each DNA sample?
- How expensive is the RE?
In this lab, you will use the NEB Cutter program to identify REs sites in plasmid sequences. (NEB Cutter is provided by New England Biolabs, a commercial supplier of REs.)
You will recall that plasmids are supercoiled circles. Digestion with a RE opens up a plasmid and relaxes its structure. (Without RE digestion, the apparent sizes of plasmids on agarose gels are unreliable.) The plasmids that we are using for our experiments are complex plasmids based on pYES2.1 (5886 bp). Search the results for REs that will generate clearly distinguishable restriction fragments from your plasmids. It is highly recommended that you select the same RE for all three digests! Since the plasmids are based on pYES2.1, it would not be surprising to observe some common restriction fragments in those digests.
Handling restriction endonucleases in the laboratory
The REs that we are using in the lab are highly purified (and expensive!) proteins that have been purified from recombinant bacteria. Like all enzymes, each RE functions optimally under a defined set of reaction conditions, including temperature, pH, and the concentrations of metal ions and salts. The manufacturer of our REs has developed buffers that support high levels of activity for more than 200 REs. Each buffer contains 0.1 mg/mL bovine serum albumin (BSA), an abundant protein from cow serum, which helps to stabilize denaturation-prone REs and to prevent nonspecific absorption of REs to test tubes and tips.
Like all enzymes, REs are subject to spontaneous denaturation, so REs need to be handled with care. (By comparison, DNA is an exceptionally stable molecule.) The rate of protein de- naturation increases with temperature and at air/water interfaces. Some simple precautions will minimize denaturation. Follow these simple rules when you prepare the restriction digests:
- Use the recommended buffer for a particular RE.
- Keep the reactions on ice until the incubation begins.
- Be careful not to introduce bubbles. Do not use the vortex mixer.
- Add the RE last, after the other components of the reaction mixture have been assembled.