Southern blotting

Developed by Edwin Mellor Southern (1938–) in 1975, Southern blotting is a procedure that uses the processes of gel electrophoresis and DNA probes to obtain more information about a gene, namely the size of the fragment that it resides on after genomic DNA is exposed to a specific restriction enzyme.

When genomic DNA is cut using a restriction enzyme, the result is fragments of varying length. When this cut DNA is separated using gel electrophoresis, and subsequently stained using ethidium bromide, the result is not a series of distinct bands, but rather a smear (мазок) of DNA fragments. The Southern blot transfers the fragmented DNA in the agarose gel to a nitrocellulose membrane using either pressure or a vacuum so that it can be probed using a DNA probe that has been labeled with ³²P, a radioactive phosphorous isotope. A DNA probe is prepared using a short stretch of nucleotides (oligonucleotides). The probe may be generated from a cloned and sequenced similar gene in another species, or from a cloned gene within the same gene family.

There are several important steps in the Southern blot procedure. Samples are placed in an agarose gel, which typically contains a high concentration of agarose, to provide adequate separation of the fragments. Once the gel electrophoresis process is complete, the gel is exposed to a sodium hydroxide solution that denatures the double helix structure of the DNA into a single-stranded molecule. Single-stranded DNA (ssDNA) is necessary to allow binding of the nucleic acid probe later in the procedure. The gel is then transferred to a piece of nitrocellulose paper using either pressure or a vacuum.

Once the transfer is complete, the membrane is placed in a special oven to permanently bind the DNA fragments to the membrane. The probe is then placed in a solution and applied to the prepared membrane.

The environmental conditions under which the probe is applied to the membrane will determine how effective the probe will be in binding to its target DNA sequences.

Low stringent ( строгий) conditions, which typically consist of high salt concentrations and low temperatures, will allow the probe to bind to DNA fragments that are not quite a perfect match for the probe. This is especially useful if the researcher is looking for anything similar to the probe, or is using a nucleotide sequence in the probe which is derived from an organism that is evolutionarily distant from the species being studied.

High stringency involves high temperatures and low salt concentrations. Under these conditions the probe will only bind to those fragments on the DNA that are complementary to the nucleotide sequence of the probe.

The excess probe is then washed off and the nitrocellulose membrane is placed with a piece of photographic film. When developed, the film will provide an indication of those areas of the membrane where the probe bound to a DNA fragment.

Southern blots are also useful in determining whether a gene exists in multiple copies in the genome.

There are several variations of the Southern blot.

The Northern blot (originally called a “reverse-Southern” blot) separates RNA using gel electrophoresis and then probes the membrane with an RNA (sometimes DNA) probe. Northern blots are useful in studying patterns of gene expression, although they have been widely replaced by the use of microarrays.

The Western blot is used to study the proteins in a cell or tissue. Western blot uses antibodies to indicate the position of a protein on the membrane.

Biophysical Methods:

electron paramagnetic resonance (EPR)

nuclear magnetic resonance (NMR)

scanning calorimetry,

various types of spectroscopy (e.g. spectrophotometry, mass spectroscopy, fluorescence spectroscopy).

Biotechnological methods:

methods of cell engineering, genetic engineering

In the genetic many other methods analysis are used:

ontogenetic,

immunogenetic,

comparative morphological,

comparative biochemical methods,

various mathematical methods, etc.