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Molecular Basis of Heredity: Part 4. Gene Identification and Tests

Author(s): Raye L. Alford, PhD

Southern Analysis

Southern analysis permits the detection of mutations and polymorphisms in genes because changes in the sequence of genes result in changes in restriction patterns. In Southern analysis, genomic DNA is isolated from a patient’s cells and digested with an enzyme. The resulting DNA fragments are run on an agarose gel for separation according to size. To stabilize the DNA in its electrophoretic separation pattern, the DNA is hybridized (affixed) to a nylon membrane. A labeled DNA probe (short sequence of DNA tagged isotopically or chemically) specific for the gene of interest is mixed with the membrane under conditions that allow the probe to find and bind to (hybridize with) its complementary DNA on the membrane, resulting in a characteristic banding pattern for the normal gene upon isotopic or chemical signal detection. This approach can be used to analyze a particular nucleotide change if the change occurs at the site of action by a restriction enzyme and alters the ability of the enzyme to cut the DNA. This approach also can detect alterations in genes that disrupt the regular banding pattern because of deletions or other rearrangements in the gene that span restriction sites. Southern analysis also can be effective in identifying changes in the size of repetitive elements, such as triplet repeats that are associated with disease. In many cases, the changes in size of the gene associated with expansion of the repeat sequence is large enough to be seen on Southern analysis as a change in the size of the band associated with the gene. In cases of smaller repeat expansions a different type of gel substance, called polyacrylamide, is used because it is better able to separate smaller sized DNA fragments.

In the image on the slide, genomic DNA is digested with a restriction enzyme and separated by gel electrophoresis according to fragment size. The DNA is then transferred from the gel to a nylon membrane and hybridized with a radioactively labeled probe unique to the sequence of interest. In this case, the probe is complementary to the beta-globin gene and is designed to detect the mutation associated with sickle cell disease, which interrupts a restriction enzyme site. The sickle cell-associated genes remain undigested by the enzyme while the normal gene sequence is cut by the enzyme into a smaller piece of DNA. In the lane labeled A, an individual with two normal copies of the beta-globin gene was analyzed. The size of the fragment seen on the autoradiograph is that of the digested DNA, indicating two normal alleles and no sickle cell-associated allele. In the column labeled S, an individual with sickle cell disease and two copies of the sickle cell gene was analyzed. The size of the fragment seen is larger than the fragment in column A, indicating that the restriction enzyme failed to cut the DNA due to the presence of two copies of the sickle cell-associated mutation. In the column labeled AS, an individual with one copy of the normal gene and one copy of the sickle cell-associated gene was analyzed. Two bands are seen: one for the digested smaller allele on the bottom; and, one for the uncut sickle cell-associated allele on the top. This individual is a carrier for sickle cell disease.