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Southern Blot

Southern Blotting The first blotting technique to be devised is known as Southern blotting after its originator E. M. Southern.

 

This technique is capable of detecting a single specific restriction fragment in the highly complex mixture of fragments produced by cleavage of the entire human genome with a restriction enzyme. In such a complex mixture, many fragments will have the same or nearly the same length and thus migrate together during electrophoresis. Even though all the fragments are not separated completely by gel electrophoresis, an individual fragment within one of the bands can be identified by hybridization to a specific DNA probe. To accomplish this, the restriction fragments present in the gel are denatured with alkali and transferred onto a nitrocellulose filter or nylon membrane by blotting.

This procedure preserves the distribution of the fragments in the gel, creating a replica of the gel on the filter, much like the replica filter produced from clones in a library. (The blot is used because probes do not readily diffuse into the original gel.) The filter then is incubated under hybridization conditions with a specific radiolabeled DNA probe, which usually is generated from a cloned restriction fragment. The DNA restriction fragment that is complementary to the probe hybridizes, and its location on the filter can be revealed by autoradiography.

 



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