E.M. Southern invented the technique of southern blotting. This method is useful in molecular biology. In this method, hybridisation analysis is performed by transferring the DNA from a gel to a membrane. The DNA is cleaved using suitable restriction enzymes and made to run on a gel. Then it is treated with sodium hydroxide that denatures the DNA to give a single strand.

Principle of Southern Blotting

Southem blotting is a hybridisation technique used for identifying particular size of DNA from a mixture of other similar molecules. This technique relies on the principle of separating DNA fragments by gel electrophoresis and identifying them by labelled probe hybridisation.

The DNA fragments are separated based on their size and charge during electrophoresis. The separated DNA fragments are transferred on nylon membrane, and the desired DNA is detected using specific DNA probe complementary to the desired DNA. A hybridisation probe is a short (100500bp ), single stranded DNA labelled with a marker so that it can be detected after hybridisation.

Procedure

In the method of southern blotting, the DNA is transferred from a gel to a membrane for hybridisation analysis. Then the DNA is cut with suitable restriction enzymes and run on a gel. On treating with NaOH, the DNA undergoes denaturation to yield a single strand.

The DNA is transferred from agarose gel to the membrane by capillary action. The gel is placed on a filter paper saturated with buffer, and the nitrocellulose membrane is placed above the gel and covered with 2-3 layers of dry filter paper towel (figure). The buffer reaches the top papers by flowing through the gel and membrane. While flowing it carries the DNA fragment with it, but DNA is retained on the paper as it cannot pass through the membrane. This membrane with trapped DNA is exposed overnight to a solution containing the radiolabelled cDNA probe. The binding of probe to its complementary sequence is detected by autoradiography.

Applications

The technique of southern blotting has the following applications:

1. It is used to detect DNA in the sample.
2. DNA fingerprinting (an example of southern blotting) is used for paternity testing, criminal identification, and victim identification.
3. It is used to isolate and identify the desired genes.
4. It is used in restriction fragment length polymorphism.
5. It is used to identify mutation or gene rearrangement in the DNA sequence.
6. It is used in diagnosing diseases caused by genetic defects.
7. It is used to identify infectious agents.