The Southern Blotting Technique

The Southern blotting is a laboratory method used to detect specific DNA molecules from many other DNA molecules. This technique was named after its inventor, Edward Southern who developed it at Edinburgh University in 1970s. Southern blotting combines transfer of electrophoresis-separated DNA fragments to a filter membrane and subsequent fragment detection by probe hybridisation. As a lab procedure, Southern blots can be used to analyse an organism's total DNA, also known as its genome, in order to identify a specific sequence of interest.

DNA samples are cut with restriction enzymes and loaded on agarose gel for electrophoresis. After running the gel electrophoresis, DNA is separated according to size and visualised by staining, photography in UV light (when large DNA molecules are cut by restriction endonucleases, a smear is seen rather than distinct bands).

DNA is denatured (rendered single stranded) by placing DNA gel into an alkaline solution (NaOH). Now, gel is placed on sponge wick.

Transfer of single stranded DNA fragments to DNA-binding filter is facilitated using buffer solution or salt solution system. DNA binding filter, paper towels and weight are placed on gel. Buffer passes upward by capillary action, transferring DNA fragments to filter.

After DNA is transferred to the filter, treat the filter with UV light to permanently attach the transferred DNA to the filter. (Alternatively, you can bake the filter in the oven at about 80°C for 2 hours.)

Filter placed in heat-sealed bag with solution containing radioactive probe. A probe is a fragment of labeled, single-stranded nucleic acid that is complementary to the DNA of interest.

Filter is washed to remove excess probe

Those fragment to which the probe hydrogen bonds become radio-active and are readily detected by autoradiography. In this technique, a sheet of photographic film (X-ray film) is placed over the filter.

When developed, bands appear wherever a radioactive fragment is located because the energy released by the isotope causes the formation of dark-silver grains.

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