Blotting technique is an extremely powerful tool for analyzing gene structure and used to study gene expression, once cloned cDNA is isolated. There are three important types of blotting techniques are: 1. Southern Blotting 2. Northern Blotting 3. Western Blotting.
Technique # 1. Southern Blotting:
Developed by E.M. Southern, the technique of Southern blotting is one of the most important methods used in molecular biology. In Southern blotting, DNA is transferred from a gel to a membrane for hybridization analysis. In this technique, the DNA is cut with suitable restriction enzymes and run on a gel. Treatment with NaOH denatures the DNA to form single strand.
The transfer of DNA from agarose gel to the membrane is performed by capillary action. The gel is placed above the buffer saturated filter paper. The nitrocellulose membrane is placed above the gel and covered by 2-3 layers of dry filter paper towel. A flow of buffer occurs through the gel and membrane to the top papers.
The flow carries the DNA fragment with it. However, DNA cannot pass through the membrane and is fixed firmly to the paper. The membrane, to which the DNA is trapped, is then exposed overnight to a solution containing the radio-labelled cDNA probe. The binding of probe to its complementary sequence is then detected by autoradiography (Fig. 13.3(a)).
Significance:
Southern blotting is useful for detecting major gene arrangements. This technique plays important role in DNA finger print, identification of novel gene, identification of structurally related genes in the species etc.
Technique # 2. Northern Blotting:
Northern blotting is a technique used to analyse RNA. In northern blot, RNA is transferred from agarose gel to nitrocellulose paper for hybridization analysis. Total cellular RNA or poly (A) RNA, is separated by size on an agarose gel. The RNA molecules in the gel can be transferred to nitrocellulose or nylon membrane.
The RNA molecules are separated on agarose gel containing formaldehyde or dimethylsulfoxide. The formaldehyde is used to alter secondary structure of RNA molecules. Nitrocellulose filter paper binds strongly to denatured RNA, but not with RNA having secondary structure. The nitrocellulose paper becomes rective after treating with aminobenzyl oxymethyl.
After blotting RNA to chemically reactive paper, they are hybridized to radiolabeled DNA probe. Autoradiography is then carried out to locate RNA bands that are complementary to the probe (Fig. 13.3(b)).
Significance:
Northern blotting is useful in the identification of a particular gene expression in a tissue or cell type. It is useful in cDNA cloning because the size of a specific mRNA can be compared with the size of cloned cDNA.
Technique # 3. Western Blotting:
Western blotting is associated with the transfer of proteins from acrylamide gel to nitrocellulose or nylon membrane. This technique is useful in identification of a particular gene product. Any target protein can be identified by immunological screening method.