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Blotting techniques are very widely used analytical tools for the specific identification of desired DNA or RNA fragments from thousands of molecules. Blotting refers to the process of immobilization of sample nucleic acids or solid support. The blotted nucleic acids are then used as targets in the hybridization experiments for their specific detection.

Types of blotting techniques

The most commonly used blotting techniques are Southern blotting for DNA, Northern blotting for RNA, Dot blotting, Colony and plaque hybridization.

The scientist Ed Southern developed the southern blotting in 1975. The names Northern blotting and Western blotting are laboratory jargons, which are now accepted. Western blotting involves the transfer of protein blots and their identification by using specific antibodies.

Southern blotting

Southern blotting technique is the first nucleic acid blotting procedure developed in 1975 by Southern. The genomic DNA isolated from cells and tissues is digested with one or more restriction enzymes. The mixture is loaded into a well in an agarose or polyacrylamide gel and then subjected to electrophoresis. DNA, being negatively charged migrates towards the anode the smaller DNA fragments move faster.

The separated DNA molecules are denatured by exposure to a mild alkali and transferred to nitrocellulose or nylon paper. This results in an exact replica of the pattern of DNA fragments on the gel. The DNA can be annealed to the paper on exposure to heat. The nitrocellulose or nylon paper is then exposed to labeled cDNA probes. These probes hybridize with complementary DNA molecules on the paper.

The paper after thorough washing is exposed to X-ray film to develop autoradiograph. This reveals specific bands corresponding to the DNA fragments recognized by cDNA probe.