Southern Blotting

In 1975 Ed Southern developed an ingenious technique, now known as Southern blotting, which can be used to detect specific genes (Fig. 7.12). Genomic DNA is isolated and digested with one or more restriction endonucleases. The resultant fragments are separated according to size by agarose gel electrophoresis. The gel is soaked in alkali to break hydrogen bonds between the two DNA strands and then transferred to a nylon membrane. This produces an exact replica of the pattern of DNA fragments in the agarose gel. The nylon membrane is incubated with a cloned DNA fragment tagged with a radioactive label (page 143). The gene probe is heated before adding to the nylon membrane to make it single-stranded so it will base pair, or hybridize, to its complementary sequences on the nylon membrane. As the gene probe is radiolabeled, the sequences to which it has hybridized can be detected by autoradiography.

Mutations that change the pattern of DNA fragments—for instance, by altering a restriction endonuclease recognition site or deleting a large section of the gene—can easily be detected by Southern blotting. This technique is therefore useful in determining whether an individual carries a certain genetic defect or if a fetus is homozygous for a particular disorder. All that is needed is a small DNA sample from white blood cells or, in the case of a fetus, from the amniotic fluid in which it is bathed, or by removing a small amount of tissue from the chorion villus that surrounds the fetus in the early stages of pregnancy.

Forensic laboratories use Southern blotting to generate DNA fingerprints from samples of blood or semen left at the scene of a crime. A DNA fingerprint is a person-specific Southern blot. The gene probe used in the test is a sequence that is repeated very many times within the human genome—a microsatellite sequence (page 100). Everyone carries a different number of these repeated sequences, and because they lie adjacent to each other on the chromosome they are called VNTRs (variable number tandem repeats). When genomic DNA is digested with a restriction endonuclease and then analyzed by Southern blotting, a DNA pattern of its VNTRs is produced. Unless they are identical twins, it is extremely unlikely that two individuals will have the same DNA fingerprint profile. It has been estimated that if eight restriction endonucleases are used, the probability of two people who are not identical twins generating the same pattern is one in 1030.

A special type of Southern blot, called a zoo blot, can be used to reveal genes that are similar in different species. Such genes, which have been conserved through evolution, are likely to code for crucial proteins. A probe generated from a genomic DNA library is used to probe genomic DNA from many different species. A probe that hybridizes with DNA from a number of species is likely to represent all or part of such a conserved gene.

DNA transfers from the gel to the nylon membrane as the salt solution passes through the gel and membrane and into the paper towels nylon membrane agarose gel

DNA transfers from the gel to the nylon membrane as the salt solution passes through the gel and membrane and into the paper towels nylon membrane agarose gel nylon membrane incubated with radioactive cDNA probe ( )

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