Locating DNA Fragments with Southern Blotting and Probes

If a 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 3.2 billion base pairs, would generate more than 12 million fragments when cut by this restriction enzyme. When separated by electrophoresis and visualized, this large set of fragments would appear as a continuous smear on the gel because of the presence of so many fragments of differing sizes. Usually, researchers are interested in only a few of these fragments, perhaps those carrying a specific gene. How do they 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 the probe will pair only with the bases on a complementary sequence, so, if suitably labeled, the probe can be used to locate a specific gene or other DNA sequence. To use a probe, a researcher first cuts the DNA into fragments by using one or more restriction enzymes and then separates the fragments with gel electrophoresis (Figure 14.3). Next, the separated fragments must be denatured (the two strands separated) and transferred to a permanent solid medium, such as a nitrocellulose or nylon membrane. Southern blotting (named after Edwin M. Southern) is one technique used to transfer the denatured, single-stranded fragments from a gel to a membrane.

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Figure 14.3: Southern blotting and hybridization with probes can locate a few specific fragments in a large pool of DNA.

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After the single-stranded DNA fragments have been transferred, the membrane is placed in a hybridization solution containing a labeled probe (see Figure 14.3). The probe binds to (hybridizes with) any DNA fragments on the membrane that bear complementary sequences. Often, a probe binds to only a part of the DNA fragment, so the DNA fragment may contain sequences not found in the probe. The membrane is then washed to remove any unbound probe; a biochemical method then reveals the presence of the bound probe. Thus, Southern blotting can reveal the presence of a specific DNA fragment in a genome or sample of DNA.

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

CONCEPTS

Labeled probes, which are sequences of RNA or DNA that are complementary to the sequence of interest, can be used to locate individual genes or DNA sequences. Southern blotting can be used to transfer DNA fragments from a gel to a membrane.