Nucleic Acid Hybridization – Blotting Techniques

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Blotting techniques are widely used analytical tools for the specific identification of desired DNA or RNA fragments from larger number of molecules. Blotting refers to the process of immobilization of sample nucleic acids or solid support (nitrocellulose or nylon membranes.) The blotted nucleic acids are then used as target in the hybridization experiments for their specific detection.

Types of Blotting Techniques

Southern Blotting:

The transfer of DNA from agarose gels to nitrocellulose membrane.

Northern Blotting:

The transfer of RNA to nitrocellulose membrane.

Western Blotting:

Electrophoretic transfer of Proteins to nitrocellulose membrane.

Southern Blotting Techniques – DNA

The transfer of denatured DNA from Agarose gel to Nitrocellulose Blotting or Filter Paper technique was introduced by Southern in 1975 and this technique is called Southern Blotting Technique.

Steps

The transfer of DNA from agarose gel to nitrocellulose filter paper is achieved by Capillary Action.

A buffer Sodium Saline Citrate (SSC) is used, in which DNA is highly soluble, it can be drawn up through the gel into the Nitrocellulose membrane.

By this process ss-DNA becomes ‘Trapped’ in the membrane matrix.

This DNA is hybridized with a nucleic acid and can be detected by autoradiography.

Autoradiography – A technique that captures the image formed in a photographic emulsion due to emission of light or radioactivity from a labelled component placed together with unexposed film.

Northern Blot

It was found that RNA is not binding to cellulose nitrate. Therefore, Alwin et al. (1979) devised a procedure in which RNA bands are transferred from the agarose gel into nitrocellulose filter paper. This transfer of RNA from gel to special f ilter paper is called Northern Blot hybridization. The filter paper used for Northern blot is Amino Benzyloxymethyl Paper which can be prepared from Whatman 540 paper.

Western Blot

Refers to the electrophoretic transfer of proteins to blotting papers. Nitrocellulose filter paper can be used for western blot technique. A particular protein is then identified by probing the blot with a radio-labelled antibody which binds on the specific protein to which the antibody was prepared.

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