Nucleic Acid Hybridization - Blotting Techniques

Blotting is a family of techniques built around one shared idea: take nucleic acids (or protein) that are still mixed together on a gel, and transfer them onto a solid membrane support - nitrocellulose or nylon - where they become 'immobilised' and can then be probed individually for a specific target sequence or molecule through a hybridization reaction. Southern blotting, named after its inventor E.M. Southern (1975), is the original version: DNA that has already been separated by size on an agarose gel is denatured with alkali, then drawn by capillary action, through an SSC (sodium saline citrate) buffer, up out of the gel and onto a nitrocellulose membrane, where the resulting trapped single-stranded DNA is hybridised with a labelled probe and finally visualised by autoradiography - detecting the pattern of radioactivity left by the bound probe on photographic film. Northern blotting, worked out later by Alwine and colleagues (1979), applies the same basic logic to RNA instead of DNA - RNA does not bind ordinary nitrocellulose well, so a different membrane, amino-benzyloxymethyl paper (made from Whatman 540 paper), is used instead. Western blotting is different again in what it moves: it electrophoretically transfers proteins (not nucleic acids) onto a nitrocellulose membrane, where a specific protein of interest is then identified using a radio-labelled antibody that binds only to that protein. Beyond basic molecular biology, these hybridisation-based tools also underpin plant-pathogen diagnostics: ELISA (Enzyme-Linked Immuno Sorbent Assay) identifies pathogen spe …