Southern+Blotting


 * Southern Blot Technique **

Southern Blotting is a traditional technique used in Molecular Biology which employs the use of Gel Electrophoresis and hybridization to determine the presence of a particular segment of DNA. Southern Blotting has many uses which include; determining the molecular weight of a DNA fragment, genome mapping of a given species, detecting genetic diseases in organisms by identifying specific sequences of DNA and manufacturing chromosomal linage maps. (Davidson)
 * Technique/Applications**


 * Protocol**

The first step is to prepare the DNA mixture by using endonucleases (enzymes the sever the phosphodiester bond with a polynucleotide chain). These cut the DNA sample into smaller fragments which are then run through an agarose gel using Gel Electrophoresis to separate the DNA fragments by size. These double stranded fragments are then denatured into single strands. The single strands are transferred from the gel on to a nitrocellulose membrane via “blotting,” which is where this technique’s name is derived from. The blotting of the gel is set on a sponge in an alkali solution. Buffer, which also denatures the DNA fragments, is sucked through the gel and on to the sheet by paper towels (Figure 1). This nitrocellulose membrane is treated with probes which are specifically chosen to contain a complimentary message which hybridizes to the target strand of DNA. These probes oftentimes contain some form of label, whether it is a radioactive atom or a fluorescent dye, which can be detected by various equipment. (Anonymous)

Figure 1: Southern Blotting Technique http://wiki.biomine.skelleftea.se/biomine/molecular/index_20.htm

For a brief but instructive video, please check out this!

This technique was invented by Edward Southern at Edinburgh University in 1975 and was named after him. Due to the significance of this technique, similar techniques have been named in his honor; Northern Blotting (used to study gene expression by the detection of RNA) and Western Blotting (protein detection). Though Southern Blotting is an effective process, it has been around for nearly 45 years and is considered time-consuming. Some newer techniques have been invented such as long-range PCR that are slowly replacing Southern Blotting. New, dig-labelled probes are now being used which is non-radioactive and detected by fab-fragments (Solanas).
 * History**

In the Cecchini et al article, the researchers used the Southern Blot technique to detect a bacteria, //candidatus arthromitus//, in the intestines of trout. This bacteria has caused gastroenteritus in rainbow trout and has significantly decreased their numbers resulting in large economic losses in areas that rely on the rainbow trout. Southern blotting made it possible to detect the DNA band of //c. arthromitus//.
 * Articles**

In the Chen et al. article, they have found that by using cgg repeat primed PCR, they can obtain a better result quicker than southern blot. They obtained the same information that they would have with in using the Southern Blot technique including all of the full mutations. What this provides is that the technique will now require less samples than required by southern blotting technique.

Anonymous, 1. (n.d.). //Southern blotting//. Retrieved from http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/DNA-RNA-Purification-Analysis/Nucleic-Acid-Gel-Electrophoresis/Southern-Blotting.html
 * References**

Cecchini, F., Lacumen, L., Fontanot, M., Comi, G., & Manzano, M. (2012). Identification of the unculturable bacteria //candidatus arthromitus// in the intestinal content of trouts using dot blot and Southernblot techniques. //Veterinary Microbiology//, //156//(3-4), 389-394.

Chen, L., Hadd, A., Sah, S., Filipovic-Sadic, S., Krosting, J., Sekinger, E., Pan, R., & Hagerman, P. (2010). An information-rich cgg repeat primed PCR that detects the full range of fragile x expanded alleles and minimizes the need for Southernblot analysis. //Journal of Molecular Diagnostics//, //12//(5), 589-600.

Davidson. (2001). //Southern blot method//. Retrieved from http://www.bio.davidson.edu/courses/genomics/method/southernblot.html

Solanas, M., & Escrich, E. (1997). An improved protocol to increase sensitivity of southernblot using dig-labelled dna probes. //Journal of Biochemical and Biophysical Methods//, //35//(3), 153-159.