A Southern blot is a method used in molecular
biology for detection of a specific DNA sequence
in DNA samples.
Southern blotting combines transfer of electrophoresis-separated
DNA fragments to a filter membrane and subsequent
fragment detection by probe hybridization.
The method is named after its inventor, the
British biologist Edwin Southern.
Other blotting methods (i.e., western blot,
northern blot, eastern blot, southwestern
blot) that employ similar principles, but
using RNA or protein, have later been named
in reference to Edwin Southern's name.
As the label is eponymous, Southern is capitalised,
as is conventional for proper nouns.
The names for other blotting methods may follow
this convention, by analogy.
Restriction endonucleases are used to cut
high-molecular-weight DNA strands into smaller
fragments.
The DNA fragments are then electrophoresed
on an agarose gel to separate them by size.
If some of the DNA fragments are larger than
15 kb, then prior to blotting, the gel may
be treated with an acid, such as dilute HCl.
This depurinates the DNA fragments, breaking
the DNA into smaller pieces, thereby allowing
more efficient transfer from the gel to membrane.
If alkaline transfer methods are used, the
DNA gel is placed into an alkaline solution
(typically containing sodium hydroxide) to
denature the double-stranded DNA.
The denaturation in an alkaline environment
may improve binding of the negatively charged
thymine residues of DNA to a positively charged
amino groups of membrane, separating it into
single DNA strands for later hybridization
to the probe (see below), and destroys any
residual RNA that may still be present in
the DNA.
The choice of alkaline over neutral transfer
methods, however, is often empirical and may
result in equivalent results.[citation needed]
A sheet of nitrocellulose (or, alternatively,
nylon) membrane is placed on top of (or below,
depending on the direction of the transfer)
the gel.
Pressure is applied evenly to the gel (either
using suction, or by placing a stack of paper
towels and a weight on top of the membrane
and gel), to ensure good and even contact
between gel and membrane.
If transferring by suction, 20X SSC buffer
is used to ensure a seal and prevent drying
of the gel.
Buffer transfer by capillary action from a
region of high water potential to a region
of low water potential (usually filter paper
and paper tissues) is then used to move the
DNA from the gel onto the membrane; ion exchange
interactions bind the DNA to the membrane
due to the negative charge of the DNA and
positive charge of the membrane.
The membrane is then baked in a vacuum or
regular oven at 80 degree C for 2 hours (standard
conditions; nitrocellulose or nylon membrane)
or exposed to ultraviolet radiation (nylon
membrane) to permanently attach the transferred
DNA to the membrane.
The membrane is then exposed to a hybridization
probe, a single DNA fragment with a specific
sequence whose presence in the target DNA
is to be determined.
The probe DNA is labelled so that it can be
detected, usually by incorporating radioactivity
or tagging the molecule with a fluorescent
or chromogenic dye.
In some cases, the hybridization probe may
be made from RNA, rather than DNA.
To ensure the specificity of the binding of
the probe to the sample DNA, most common hybridization
methods use salmon or herring sperm DNA for
blocking of the membrane surface and target
DNA, deionized formamide, and detergents such
as SDS to reduce non-specific binding of the
probe.
After hybridization, excess probe is washed
from the membrane (typically using SSC buffer),
and the pattern of hybridization is visualized
on X-ray film by autoradiography in the case
of a radioactive or fluorescent probe, or
by development of colour on the membrane if
a chromogenic detection method is used.
Hybridization of 
the probe to 
a specific DNA fragment on the filter membrane
indicates that this fragment contains DNA
sequence that is complementary to the probe.
The transfer step of the DNA from the electrophoresis
gel to a membrane permits easy binding of
the labeled hybridization probe to the size-fractionated
DNA.
It also allows for the fixation of the target-probe
hybrids, required for analysis by autoradiography
or 
other 
detection methods.
Southern blots performed with restriction
enzyme-digested genomic DNA may be used to
determine the number of sequences (e.g., gene
copies) in a genome.
A probe that hybridizes only to a single DNA
segment that has not been cut by the restriction
enzyme will produce a single band on a Southern
blot, whereas multiple bands will likely be
observed when the probe hybridizes to several
highly similar sequences (e.g., those that
may be the result of sequence duplication).
Modification of the hybridization conditions
(for example, increasing the hybridization
temperature or decreasing salt concentration)
may be used to increase specificity and decrease
hybridization of the probe to sequences that
are less than 100% similar.
Southern transfer may be used for homology-based
cloning on the basis of amino acid sequence
of the protein product of the target gene.
Oligonucleotides are designed that are similar
to 
the target sequence.
The oligonucleotides are chemically synthesised,
radiolabeled, and used 
to screen a DNA library, or other collections
of cloned DNA fragments.
Sequences that hybridize with the hybridization
probe 
are further analysed, for example, to obtain
the full length sequence of the targeted gene.
Southern blotting can also be used to identify
methylated sites 
in particular genes.
Particularly useful are the restriction nucleases
MspI and HpaII, both of which recognize and
cleave within the same sequence.
However, HpaII requires that 
a C within that site be methylated, whereas
MspI cleaves only DNA unmethylated at that
site.
Therefore, any methylated sites within a sequence
analyzed with a 
particular 
probe 
will be cleaved by the former, but not 
the latter, enzyme.
