Provides information on the biochemical technique known as Southern Blotting
Overview of Southern Blotting
Southern hybridization is the identification of DNA sequences by separation on gels and the binding of specific complementary probe sequences by base pairing.The DNA to be probed is digested by restriction enzymes and pieces separated by agarose gels.
The DNA that is denatured is transferred to membrane (nylon) and X-linked to the membrane.
Labelled probe is hybridized to DNA.
Hybridization is detected by the autoradiogram, fluorescence, or enzymatic reaction.
Properties of Southern Blotting
R.E Digest Bands appear as a smear because there are so many. Can be used for population genetics or e.g. if looking for the presence of Cystic Fibrosis (C.F), you can look for the complementary sequence of GCCAT which is CGGTA.
There are many forms of restriction sites as every person is different we have different restriction sites with different fragment lengths, so when they pass through the agarose gel the larger fragments get stuck and the smaller fragments flow to the bottom.
In the pre-hybridisation step, we place the membrane in hybridisation solution to ensure that the probe does not bind to the wrong sequence.
Incubate at 65C overnight as to allow the probe to anneal with the single stranded restriction fragments that are connected to the membrane.