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 agarose gel electrophoresis for size separation of DNA with methods to transfer the size-separated DNA to a filter membrane for probe hybridization. The method is named after its inventor, the British biologist Edwin Southern.^[1] Other blotting methods (i.e., 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. Molecular biology is the study of biology at a molecular level. ... Digital image of 3 plasmid restriction digests run on a 1% w/v agarose gel, 3 Volts/cm, stained with ethidium bromide. ... It has been suggested that Electrophoretic mobility be merged into this article or section. ... In molecular biology, a hybridization probe is a fragment of DNA of variable length (usually 100-1000 bases long), which is used to detect in DNA or RNA samples the presence of nucleotide sequences that are complementary to the sequence in the probe. ... A biologist is a scientist devoted to and producing results in biology through the study of organisms. ... Sir Edwin Southern (born 1938) is a 2005 Lasker Award-winning molecular biologist. ... In molecular biology and genetics, a blot is a method of transferring proteins, DNA or RNA, onto a carrier (for example, a nitrocellulose PVDF or nylon membrane). ... Picture of a western blot with 5 vertical lanes A western blot (a. ... The northern Blot is a technique used in molecular biology research to study gene expression. ... Southwestern blotting Southwestern blotting, played along the lines of Southern blotting (created by Edwin Southern), involves characterizing proteins that bind to DNA. The proteins undergo gel electrophoresis and are subsequently transferred to nitrocellulose membranes similar to other types of blotting. ...

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, 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 sodium hydroxide) to denature the double-stranded DNA. The denaturation in an alkaline environment provides for improved binding of the negatively charged DNA to a positively charged membrane, separates 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.
5. A sheet of nitrocellulose (or, alternatively, nylon) membrane is placed on top (or below, depending on the direction of the transfer) of 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. 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 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 ultraviolet radiation (nylon) to permanently and 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 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.
8. After hybridization, excess probe is washed from the membrane, 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 color on the membrane if a chromogenic detection method is used.

Endonucleases are enzymes that cleave the phosphodiester bond within a nucleotide chain. ... HCL or HCl can stand for: Hardware Compatibility List Chemical symbol for hydrochloric acid, written HCl Higher Chinese Language, an academic subject in Singapore An Indian software company (previously Hindustan Computers Ltd. ... Depurination is DNA alteration in which the hydrolysis of a purine base (Adenine or Guanine) from the deoxyribose-phosphate backbone occurs. ... Sodium hydroxide (NaOH), also known as lye or caustic soda, is a caustic metallic base. ... Hybridisation is the process of combining complementary, single-stranded nucleic acids into a single molecule. ... Nitrocellulose Nitrocellulose Nitrocellulose (also: cellulose nitrate, flash paper) is a highly flammable compound formed by nitrating cellulose through, for example, exposure to nitric acid or another powerful nitrating agent. ... Nylon is a generic designation for a family of synthetic polymers first produced on February 28, 1935 by Gerard J. Berchet of Wallace Carothers research group at DuPont. ... An artificial membrane, also called a synthetic membrane, is a membrane prepared for separation tasks in laboratory and industry. ... Capillary action, capillarity, or capillary motion is the ability of a substance (the standard reference is to a tube in plants but can be seen readily with porous paper) to draw a substance up against gravity. ... Water potential of negative water solution Water potential is the tendency of water to move from one place to another. ... Ion exchange is a process in which ions are exchanged between a solution and an ion exchanger, an insoluble solid or gel. ... Note: Ultraviolet is also the name of a 1998 UK television miniseries about vampires. ... Covalent bonding is a form of chemical bonding characterized by the sharing of one or more pairs of electrons between atoms, in order to produce a mutual attraction, which holds the resultant molecule together. ... A hybridization probe is a short piece of DNA (on the order of 100-500 bases) that is denatured (by heating) into single strands and then radioactively labeled, usually with phosphorus (32P or 33P). ... Radioactivity may mean: Look up radioactivity in Wiktionary, the free dictionary. ... Fluorescence induced by exposure to ultraviolet light in vials containing various sized Cadmium selenide (CdSe) quantum dots. ... In the NATO phonetic alphabet, X-ray represents the letter X. An X-ray picture (radiograph) taken by Röntgen An X-ray is a form of electromagnetic radiation with a wavelength approximately in the range of 5 pm to 10 nanometers (corresponding to frequencies in the range 30 PHz... An autoradiograph is an image produced on a photographic film by the radiation from a radioactive substance. ...

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.

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

• Restriction fragment
• Genetic fingerprint

A DNA fragment resulting from cleavage of a DNA strand by a restriction enzyme. ... Genetic fingerprinting or DNA testing is a technique to distinguish between individuals of the same species using only samples of their DNA. Its invention by Sir Alec Jeffreys at the University of Leicester was announced in 1985. ...

External links

• Southern blot Animation