The first blotting technique to be devised is
known as Southern blotting after its originator
E. M. Southern.
This technique is
capable of detecting a single specific
restriction fragment in the highly complex
mixture of fragments produced by cleavage of the
entire human genome with a restriction enzyme.
In such a complex mixture, many fragments will
have the same or nearly the same length and thus
migrate together during electrophoresis. Even
though all the fragments are not separated
completely by gel electrophoresis, an individual
fragment within one of the bands can be
identified by hybridization to a specific DNA
probe. To accomplish this, the restriction
fragments present in the gel are denatured with
alkali and transferred onto a nitrocellulose
filter or nylon membrane by blotting.
preserves the distribution of the fragments in
the gel, creating a replica of the gel on the
filter, much like the replica filter produced
from clones in a library. (The blot is used
because probes do not readily diffuse into the
original gel.) The filter then is incubated
under hybridization conditions with a specific
radiolabeled DNA probe, which usually is
generated from a cloned restriction fragment.
The DNA restriction fragment that is
complementary to the probe hybridizes, and its
location on the filter can be revealed by