Southern Blotting

Raymond Dalgleish, Department of Genetics, University of Leicester, Leicester, UK

Copyright © 1998 Elsevier Ltd. All Rights Reserved.

Southern blotting is the transfer of DNA from an electrophoresis gel on to a dimensionally stable membrane support resulting in an hybridizable 'image' of the DNA banding pattern. Typically, restriction enzyme-digested DNA is size-fractionated by agarose gel electrophoresis. The double-stranded DNA is then denatured in situ by soaking the gel in alkali and transferred by capillary action to the membrane (usually nylon or nitrocellulose). The now single-stranded DNA is fixed to the membrane and may act as a target for hybridization with a specific nucleic acid probe. The probe, which is also single-stranded, will form hybrids with target DNA sequences on the filter to which it is complementary and, following extensive washing of the filter to remove unbound probe, specific hybridization is detected by a method dependent on the means by which the probe was labeled.

Autoradiography is used for the detection of radiolabeled probes. The sizes of the detected DNA fragments may then be determined by reference to DNA size markers on the original electrophoresis gel. These sizes may be of use in determining restriction enzyme maps of genes, revealing variations known as restriction fragment length polymorphisms (RFLPs). Southern blotting may also reveal alterations in gene structure such as insertions, deletions and rearrangements arising from recombination events.

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