Southern blot



A Southern blot is a method routinely used in molecular biology to check for the presence of a DNA sequence in a DNA sample. Southern blotting combines western blot, northern blot, southwestern blot) that employ similar principles, but using RNA or protein, have later been named in reference to Southern's name. As the technique was eponymously named, Southern blot should be capitalised, whereas northern and western blots should not.

Method

  1. Restriction endonucleases are used to cut high-molecular-weight DNA strands into smaller fragments.
  2. The DNA fragments are then electrophoresed on an agarose gel to separate them by size.
  3. 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, which depurinates the DNA fragments, breaking the DNA into smaller pieces, thus allowing more efficient transfer from the gel to membrane.
  4. If alkaline transfer methods are used, the DNA gel is placed into an alkaline solution (typically containing hybridization to the probe (see below), and destroys any residual RNA that may still be present in the DNA.
  5. A sheet of 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 on to 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.
  6. The membrane is then baked, i.e., exposed to high temperature (60 to 100 °C) (in the case of nitrocellulose) or exposed to covalently crosslink the DNA to the membrane.
  7. 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 SDS to reduce non-specific binding of the probe.
  8. After hybridization, excess probe is washed from the membrane, and the pattern of hybridization is visualized on autoradiography in the case of a radioactive or fluorescent probe, or by development of color on the membrane if a chromogenic detection method is used.

Result

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.

References

  1. ^ Southern, E.M. (1975): "Detection of specific sequences among DNA fragments separated by gel electrophoresis", J Mol Biol., 98:503-517. PMID 1195397

See also
 
This article is licensed under the GNU Free Documentation License. It uses material from the Wikipedia article "Southern_blot". A list of authors is available in Wikipedia.