Analysis of DNA by the Southern Blot technique
DNA is
applied to an agarose gel, and electrophoresis
separates the fragments of DNA according to size. The
gel is then placed atop a thin sponge
wick resting in a dish of salt solution, and a
special filter (typically nitrocellulose) is placed on
top of the gel. A stack of absorbent material (typically
paper towels) is placed on top of this stack. The absorbent
material draws the salt solution from the dish into the wick and
through the gel by capillary action, which transfers the DNA
fragments into the filter. The procedure is called a "Southern
transfer" after the scientist Eric Southern who
invented the procedure. The filter now contains the
DNA fragments in the same pattern as the gel, but is more
easily manipulated.
The filter is placed in a standard "seal-a-meal" bag, containing a solution of radioactively-labelled DNA probe [~~~~~] for a particular gene sequence. The probe binds to the filter only where a complementary DNA sequence is located. After washing to remove unbound probe, a piece of X-ray film is placed over the hybridized filter and exposed for several hours to several days. The radioactive label produces a black band on the film where it has stuck to the complementary DNA, producing an autoradiogram. If a labelled size marker has been used, the exact sizes of the fragments can be determined.
The Southern Blot
technique is useful for identifying a DNA sequence that
appears only once or twice in the genome, the typical situations
with nuclear loci. Note that in this example, the
electrophoresis gel shows a continuous "smear" of DNA of
all fragment sizes, whereas the autoradiogram identifies exactly
three fragments, of three different sizes.