Southern Blotting
Southern blotting uses probes to detect given DNA sequences in restriction fragments that have been separated by size using gel electrophoresis
(figure 9.9). Like the procedure for colony blotting, the DNA is transferred in-place (“blotted”) to a nylon membrane and the probe then added. Before
the transfer, however, the gel is soaked in an alkaline solution to denature the DNA.
Southern blotting is named after the scientist who developed the procedure, E. M. Southern. Analogous techniques involving separation of
macromolecules and probing for certain sequences or motifs have now been developed to study RNA and protein. To reflect the relationship of these
techniques to Southern blotting, they are referred to as Northern blotting and Western blotting, respectively.
The most obvious application of Southern blotting is to locate DNA sequences that have similar sequences to the one being studied, which is how
many functionally related genes have been discovered and characterized. In this case, one strand of the gene being studied is used as a probe. This
information can also be used to simplify a cloning experiment. For example, if the gene of interest is shown to be encoded on a restriction fragment
6,000 base pairs in length, then fragments of that size can be separated from the rest and cloned.
A less apparent but equally important use of the Southern blot is to distinguish different strains of a given species by detecting subtle variations in
their nucleotide sequences. Certain mutations will create, others will destroy, restriction enzymes recognition sequences at particular sites in the
genome. Thus, when genomic DNA of different strains is digested with the same restriction enzyme, each will give rise to a slightly different assortment
of restriction fragment sizes. Each variation is called a restriction fragment length polymorphism (RFLP). Southern blot hybridization is used to
selectively visualize restriction fragments that often vary in size. The probe employed is one that has already been shown by trial and error to hybridize
to fragments that demonstrate maximal differences among various strains.
Techniques Used in Southern Blotting
Southern blotting is used to locate specific sequences in restriction
fragments that have been separated by gel electrophoresis (see
figure 9.9). A blotting step transfers the sample in-place to a nylon
membrane that serves as a solid support, and procedures are
incorporated to denature the double-stranded DNA. A solution
containing the probe is then added to the membrane and incubated
under conditions that allow the probe to hybridize to
complementary sequences on the filter. Any probe that has not
bound is then washed off. If the probe was labeled with a
radioactive isotope, a process called autoradiography is used to
locate the positions of the hybridized probe. In this procedure, Xray film is placed on the filter containing the probe-bound DNA
and incubated. After the film is developed, visible black grains can
be seen at the sites where probe was located. Different methods are
used to detect probes that have been labeled with other markers.