Southern blotting


Southern blotting is a technique used in molecular biology to locate a specific base sequence within a strand of DNA.

The DNA of interest is initially digested into fragments by restriction enzymes. The digested fragments are then separated from each other in a procedure called agarose-gel electrophoresis; all DNA fragments are negatively charged and move towards the positively charged anode in electrophoresis, but the distance moved in the agarose gel corresponds to fragment size, with smaller fragments moving further than larger fragments.

Once separated, the fragments are denatured from double-stranded to single-stranded form by treatment with sodium hydroxide (NaOH). This occurs in-situ while the fragments are in the agarose gel. Once all fragments are denatured, a positively charged nylon filter and a stack of paper towels are placed atop the gel, and fragments are pulled towards the positively charged nylon by capillary action.

The nylon sheet is then placed in a solution of radio-labelled genetic probes. The probes are sequences of DNA that are complementary to the fragments, and they are radio-labelled to enable easy detection of the fragments. The probes are supplied in vast molar excess in order to favour hybridisation of the probes with the fragments over reannealment of the fragments with their original complementary partners. The concentration of NaCl in the probe solution can be adjusted to give the desired stringency of hybridisation. Excess probe is washed off by reducing NaCl concentration.

Finally, the radio-labelled fragments are exposed to X rays; the position of the fragments on the nylon sheet corresponds to black bands where X-rays have been absorbed. This is called autoradiography.