Southern Blotting (Hybridization) Technique : In 1975, Edward M. Southern developed the technique of DNA separation and its hybridization. Therefore, in his honor this technique is known as ‘Southern blotting or Southern hybridization technique’. A specific DNA fragment can be separated and identified in a heterologous population of DNA molecules on the basis of binding of DNA probe with its complementary DNA strand.
The genomic DNA is isolated from the clone and digested with restriction enzymes. The DNA fragments are separated by agarose gel electrophoresis. Different DNA bands are formed on agarose gel which represents DNA fragments of varying sizes. These fragments are transferred from gel to nylon or nitrocellulose membrane. The process of DNA transfer is called ‘blotting’.
A nitrocellulose membrane is put over the gel. Many layers of filter paper are placed over nitrocellulose membrane. This assembly is put in a container having NaOH solution. NaOH denatures DNA and results in formation of single stranded DNA. DNA fragments are transferred from gel to membrane by capillary action.
The DNA fragments are fixed to membrane by using UV radiation or baking at 80°C. The pattern of DNA bands on membrane corresponds to the position of DNA on gel. The membrane is put in solution containing radio labelled DNA probe and incubated for some time. DNA probe hybridizes complementary DNA fragments fixed on membrane. It is gently washed at 12°C and dried. The membrane is exposed through a photographic film. DNA bands formed on photographic film corresponds to the original position of DNA fragments present on agarose gel.
Southern Blotting technique involves separation and identification of a specific DNA fragment. In Northern Blotting technique the RNA is analysed rather than DNA. During southern blotting NaOH denatures DNA to form single stranded DNA which are transferred from gel to nitro cellulose membrane. In northern blotting total RNA molecules are extracted and then mRNA molecules are isolated by using oligo (dT) cellulose Chromatography. RNA samples separated are transferred to a nylon membrane.
