Southern blotting is a technique for transfer of DNA molecules from an electrophoresis gel to a nitrocellulose or nylon membrane, and is carried out prior to detection of specific molecules by hybridization probing.
In this technique, DNA is usually converted into conveniently sized fragments by restriction digestion and separated by gel electrophoresis, usually on an agarose gel. The DNA is denatured into single strands by incubation by alkali treatment.
The DNA is transferred to a nitrocellulose filter membrane which is a sheet of special blotting paper. The DNA fragments retain the same pattern of separation they had on the gel. This process is called blotting.
The nitrocellulose membrane is now: removed from the blotting stack.
The blot is incubated with many copies of a radioactive probe which is single-stranded DNA. This probe detect and identify base pairs with its complementary DNA sequence and bind to form a double-stranded DNA molecule. The probe cannot be seen but it is either radioactive or has an enzyme bound to it (e.g., alkaline phosphatase or horseradish peroxidase). This step is known as hybridization reaction.
The location of the probe is revealed by incubating it with a colorless substrate that the attached enzyme converts to a coloured product that can be seen or gives off light which will expose X-ray film. If the probe was labelled with radioactivity, it can expose X-ray film directly. The images of radioactive probe are revealed as distinct bands on the developed X-ray film.
Applications of Southern blotting
• It is used in DNA fingerprinting.
• Identify mutation, deletion and gene rearrangement.
• Prognosis of cancer and prenatal diagnosis of genetic disease.
