Analysis of Cloned DNA Sequences

Once DNA has been cloned and amplified, it needs to be analyzed and characterized. Two common ways to do this are restriction enzyme mapping (and its variation, the Southern Blot) and DNA sequencing, both of which rely upon a technique known as gel electrophoresis.

Gel electrophoresis takes advantage of the overall negative charge possessed by nucleic acids, due to the phosphates in the backbone. Because nucleic acids are negatively charged, they will migrate in an electric field from the negative electrode to the positive electrode (because like charges repel each other). If the electric field and the nucleic acids are run in a semisolid matrix (i.e. a 'gel' usually composed of agarose, a gelatin-like matrix, or polyacrylamide, a chemically cross-linked meshwork), then the nucleid acids will migrate at a speed inversely proportional to their size. In other words, large molecules will migrate slowly, because they will be held up by the gel, and small molecules will migrate quickly, because they can pass more easily through openings in the meshwork of the gel. Practically speaking, a mixture of DNA molecules can be loaded into a depression in a gel (the depression is called a 'well'), the gel is surrounded by an electrolyte solution, and electric current is passed through the gel. The fragments of DNA in the mixture will separate out according to size, and can be visualized using a DNA-specific stain, such as ethidium bromide, which binds to DNA and glows orange under UV light.

Restriction enzyme mapping entails digesting the DNA interest with various restriction enzymes, singly and in pairs. By running the various reactions on agarose gels, it is possible to determine the sizes of the digested fragments (by comparing to DNA standard fragments of known size). This in turn allows mapping of the different restriction enzyme recognition sites relative to each other. For more on this process, see the Restriction Mapping animation on the Russell CD-ROM.

Sometimes, it is necessary to visualize only a few restriction fragments out of a large set of fragments. To do this, a technique known as Southern blotting (named after the man who developed the technique) is used. Southern blotting involves taking an agarose gel after the separation of DNA fragments, denaturing the DNA (using high pH), placing the gel against a special type of filter paper (usually nylon or nitrocellulose), and using a flow of liquid to move the DNA fragments vertically out of the gel and onto the filter. The filter can then be incubated with a labeled probe, just as was done during library screening. The probe will anneal to complementary sequences, and those sequences that anneal can be visualized because of the label on the probe.

(As an aside, the same technique can also be applied to RNA. The RNA is separated on an agarose gel, transferred to a filter, and annealed to a probe. In this way, a single species of RNA can be visualized out of a mixture of thousands of different RNA molecules. This techniques, because it is an adaptation of Southern blotting, is known as Northern blotting.)