Why is restriction mapping useful

Although the names of individual REs may sound a bit like baby talk, the nomenclature is actually very systematic and is based on its biological source. Its name begins with the genus and species Eco for E. Strain RY13 of E. Restriction enzymes like EcoRI are frequently called 6-cutters, because they recognize a 6-nucleotide sequence. Of course, the distribution of nucleotides in DNA is not random, so the actual sizes of DNA fragments produced by EcoRI range from hundreds to many thousands of base pairs, but the mean size is close to bp.

DNA fragments of this length are useful in the lab, since they long enough to contain the coding sequence for proteins and are well-resolved on agarose gels. The palindromic nature of the restriction site is more obvious in the figure below. The dot in the center of the restriction site denotes the axis of symmetry. REs are sometimes referred to as molecular scissors because of their ability to generate restriction fragments that terminate with defined sequences.

We will not be generating recombinant molecules in this class, but it is important to understand their importance to modern biology. From the plasmid maps, you can see that these complex plasmids were constructed by stitching together DNA sequences from evolutionary distinct sources. 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. 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:. 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.

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 two plasmids are based on pYES2. The REs that we are using in the lab are highly purified and expensive!

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 REs.

Each buffer contains 0. The total length of the fragments in each digestion will be equal. However, because the length of each individual DNA fragment depends upon the positions of its restriction sites, each restriction site can be mapped according to the lengths of the fragments.

The information from the double-digestion is particularly useful for correctly mapping the sites. The final drawing of the DNA segment that shows the positions of the restriction sites is called a restriction map. Related Concepts 5. You have authorized LearnCasting of your reading list in Scitable. Do you want to LearnCast this session? This article has been posted to your Facebook page via Scitable LearnCast. Change LearnCast Settings.