With Southern Blotting and Probes

If a relatively small piece of DNA, such as a plasmid, is cut by a restriction enzyme, the few fragments produced can be seen as distinct bands on an electrophoretic gel. In contrast, if genomic DNA from a cell is cut by a restriction enzyme, a large number of fragments of different sizes are produced. A restriction enzyme that recognizes a four-base sequence would theoretically cut about once every 256 bp. The human genome, with 3.3 billion base pairs, would generate more than 12 million fragments when cut by this restriction enzyme. Separated by electrophoresis and stained, this large set of fragments would appear as a continuous smear on the gel because of the presence of so many fragments of differing size. Usually, one is interested in only a few of these fragments, perhaps those carrying a specific gene. How does one locate the desired fragments in such a large pool of DNA?

One approach is to use a probe, which is a DNA or RNA molecule with a base sequence complementary to a sequence in the gene of interest. The bases on a probe will pair only with the bases on a complementary sequence and, if suitably tagged with an identifying label, the probe can be used to locate a specific gene or other DNA sequence.

To use a probe, one first cuts the DNA into fragments by using one or more restriction enzymes and then separates the fragments with gel electrophoresis (I Figure 18.5). Next, the separated fragments must be denatured and transferred to a thinner solid medium (such as nitrocellulose or nylon membrane) to prevent diffusion. Southern blotting (named after Edwin M. Southern) is one technique used to transfer the denatured, single-stranded fragments from a gel to a thin solid medium.

After the single-stranded DNA fragments have been transferred, the membrane is placed in a hybridization solution of a radioactively or chemically labeled probe (see Figure 18.5). The probe will bind to any DNA fragments on the membrane that bear complementary sequences. The membrane is then washed to remove any unbound probe; bound probe is detected by autoradiography or another method for chemically labeled probes.

RNA can be transferred from a gel to a solid support by a related procedure called Northern blotting (not named after anyone but capitalized to match Southern). The hybridization of a probe can reveal the size of a particular mRNA molecule, its relative abundance, or the tissues in which the mRNA is transcribed. Here, the probe is usually an antibody, used to determine the size of a particular protein and the pattern of the protein's expression. Western blotting is the transfer of protein from a gel to a membrane.

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