15.5 : Southern Blot

Southern blotting is a technique where a labeled DNA probe hybridizes with a target DNA to detect a particular sequence within a genome.

The technique is named after Edwin Southern, the British biologist who first developed it.

The procedure follows five main steps: electrophoresis, denaturation, membrane transfer, hybridization, and visualization.

First, the genomic DNA is digested by restriction enzymes, and the resulting DNA fragments are loaded on an agarose gel and separated using gel electrophoresis.

For denaturation or separation of the double-stranded DNA into two single-stranded DNA, or ssDNA, the gel is soaked in a sodium hydroxide solution.

The gel is then placed on a sponge in a DNA neutralizing solution containing sodium chloride and tris buffer, to reset its pH to 7.0.

Next, a nylon membrane is placed on top of the gel and weighed down with a stack of paper towels. The ssDNA transfers onto the membrane through capillary action, while the high salt concentration in the neutralizing solution helps the DNA to bind.

When irradiated with UV rays, the DNA becomes covalently cross-linked to the membrane. This prevents diffusion and immobilizes the DNA bands.

The membrane is then soaked in a solution of denatured salmon sperm DNA. The solution coats the membrane and prevents any non-specific binding with the probe DNA.

The probes are short, single-stranded DNA that have a complementary sequence to the target DNA fragments. For visualization, the probes are either labeled with a radioactive phosphorus —P-32, or an enzyme that generates an easily detectable product.

When the membrane is soaked in a buffer solution containing the probes and warmed at 42 °C, the target ssDNA pairs with the complementary probe DNA to form a labeled, hybrid, double-stranded DNA.

After overnight hybridization, the membrane is washed to remove the unbound probes.

For radioactively labeled DNA, the membrane is exposed to an X-ray film for detection.

Enzyme-labeled probes can be detected by adding an appropriate substrate and visualizing the bands as the color or luminescence develops. Because the labeled probes will only bind to the DNA sequence of interest, any visible band indicates presence of the target DNA.