BLOTTING TECHNIQUES

BLOTTING TECHNIQUES

1. Southern blotting

Very specific DNA sequences can be detected using hybridization techniques with a technique known as the Southern blot. This highly sensitive technique for identifying DNA fragments by DNA: DNA hybridization is called Southern blotting, after EM Southern who devised it. This technique has very wide application in DNA analysis:
i. DNA to be hybridized is first cleaved by restriction endonucleases.
ii. Then the pieces are separated on the basis of size and charge by agarose gel electrophoresis.
iii. These fragments are transferred (adsorbed) to a nitrocellulose or nylon membrane that is laid over the gel.
iv. When this membrane is allowed to react with labeled probe (radioactive single-stranded DNA
probes), only the fragment containing the specific sequence of DNA that hybridizes the probe is detected.
v. These will hybridize with homologous DNA to form radioactive double-stranded segments, which can be detected on X-ray film.

Southern Blotting - MyBioSource Learning Center
BLOTTING TECHNIQUES – Southern blotting

2. Northern blotting

An analogous procedure for the analysis of RNA has been called northern blotting (as opposed to southern blotting). Here the RNA mixture is separated by gel electrophoresis, blotted and identified using labeled DNA or RNA probes.

Northern Blotting -

3. Western blotting (Western Blot Assay)

A similar technique for the ldentificaion of proteins (antigens) is called immunoblotting (or, in conformity with other blotting technique, western blotting). The Western blot detects antibody instead of DNA.
i. The DNA (or RNA) of a particular etiologic agent is treated with endonucleases, or the protein components of an agent are treated with proteinases to create fragments of different size.
ii. The nucleic acid or protein fragments are separated by agarose gel electrophoresis, i.e. PAGE
(sodium dodecyl sulfate-polyacrylamide gel electrophoresis).
iii. The patterns are then blotted onto nitrocellulose as in the Southern hybridization assay.
iv. The filter paper containing specific nucleic acids or proteins is then allowed to react with antiserum from a patient or animal suspected of containing antibodies against the agent. If present,
antibodies will bind to the protein or nucleic acid against which they were created, and they can
then be detected by visual methods for the detection of antibodies (e.g. enzyme-labeled probes,
fluorescent markers).

Western Blotting

Use

Diagnosis of HIV antibody

The western blot test has received wide publicity as the confirmatory test for the diagnosis of HIV antibody in sera. The specificity of the test depends on its ability to separately identify antibodies directed against different antigens of the pathogen (for example, against the surface, core and reverse
transcriptase antigens of HIV).