Question

Explain the southern hybridisation technique.

Answer 1

Southern blot hybridization refers to the detection of specific DNA fragments that have been separated by gel electrophoresis (Figure 1). After the electrophoresis the separated DNA fragments are denaturated and transferred to a nitrocellulose (or nylon) membrane sheet by blotting. In the blotting the gel is supported on a sponge in a bath of alkali solution, and buffer is sucked through the gel and the sheet by paper towels stacked on top of the nitrocellulose sheet. The buffer denaturates the DNA and transfers the single stranded fragments from the gel to the surface of the sheet, where they adhere firmly. The nitrocellulose sheet containing the bound single-stranded DNA fragments is pealed off the gel and placed in a sealed plastic bag or a box together with buffer containing labelled DNA probe specific for the target DNA sequence. The sheet is exposed to the probe under conditions favouring hybridization. After the hybridization, the sheet is removed from the bag, washed thoroughly to remove unhybridized probes and viewed using autoradiography or ultraviolet light depending on the labels used (radioactive of fluorescent). An adaptation of Southern blotting is Northern blotting, in which RNA molecules are electrophoresed through the gel instead of DNA.