Southern Blotting

What is southern blotting? Schematically present the procedure of this technique. Discuss the importance of this technique in DNA finger printing. 

Southern Blotting :-

       Southern blotting is a method developed by a molecular biologist E.M. Southern 1975 for analysing the related genes in a DNA restriction fragment. This is a procedure for transfer of denature DNA from an agarose its hybridisation with a complementary radiolabelled nucleic acid i.e. Probe.

Procedure of southern blotting :-

       Blotting is an effective and sensitive procedure to provide a physical map of restriction site within a gene located normally on a chromosomal and reveal the number of copies of gene in the genome and the degree of similarly of the gene when compared with other complementary genes. This is achieved by this method in which the following steps are performed.

(i) 1^st of all the sample DNA is subjected to mechanical shearing or restriction digestion in order to generate DNA fragments. The resultant fragments are then separated by electrophorasis by using either Agarose gel or poly acrylamide gel.

(ii) The fragments move along the gel according to their molecular weight where the smaller fragments migrate faster and furthest to the gel than the larger fragments.

(iii) The restriction fragments of DNA present in agarose gel are then denature into single stranded form by alkali treatment.

(iv) The gel is laid on the top of a buffer saturated filter paper placed on a solid support of a glass plate with  its 2 edges emerged in the buffer.

(v) A piece of nitrocellulose or nylon membrane is placed over the gel and a stack of many paper (called paper towels) are put on the nitrocellulose membrane. A weight of about 0.5 k.g. is placed on the top of the paper towels.

(vi) The buffer solution moves due to capillary action from the bottom filter paper to the top through the gel, carrying with it the denatured DNA present in the gel and transfer the single stranded DNA to the nitrocellulose membrane.

(vii) After leaving this arrangement for a few hours or overnight the weight, paper towels etc. are removed and the nitrocellulose membrane is peeled off the gel after baking at 80° for 2-3 hours to fix DNA permanently on the membrane i.e. DNA is immobilized.

(viii) The sheet containing the bound nucleic acid is placed in a sealed plastic bag together with a buffered salt solution containing radiolabelled DNA probe. The overall set up is kept for overnight.

     The overall condition favours the binding of probe with the single stranded DNA.

(ix) After the treatment the sheet is removed from the bag and washed thoroughly so that only DNA bound probe molecules remain attached and the unbound probe will washout.

(x) After drying the membrane is placed in close contact with X-ray film and incubated for a desired period to allow images due to the radioactive probes to be bound on  the film.                  

 Importance of Southern blotting in DNA finger printing :-

       The technique of DNA finger printing was developed by Alec Jeffreys in 1985. The techniques of DNA fingerprinting have found application in quite different areas. This is important in areas ike identifying cell cultures, determining family relationships, studies of animal behavior, immigration problem, identification of criminals or murderers.

       In its original form DNA finger printing of an individual is essentially a southern blot of its DNA. It is easily revealed by the following example. If 2 persons are suspected as criminal, the original criminal is easily detected by the DNA finger printing method through Southern blotting.

       After the collection of DNA sample from the site of crime is subjected to southern blotting because before visual analysis of band DNA before visual analysis of band DNA should be attached to a specialized membrane or film i.e. nitrocellulose membrane. On nitrocellulose membrane, DNA can be hybridized with radiolabelled probe. Another problem is that it is not possible to direct fixation of DNA in nitrocellulose membrane. Thus DNA strands at 1st taken into agarose gel and then transferred to nitrocellulose membrane to the southern blotting.