thumb|[[Agarose gel electrophoresis|Agarose gel]]

thumb|Tray with a stack consisting top down of a weight, paper towels, [[artificial membrane|membrane of nitrocellulose or nylon, gel, salt solution and a slab of glass.]]

thumb|Southern blot membrane after [[Nucleic acid hybridization|hybridization and rinsing.]]

thumb|Southern blot agarose gel under [[ultraviolet illumination.]]

thumb|Southern blot [[autoradiography|autoradiogram.]]

Southern blot is a method used for detection and quantification of a specific DNA sequence in DNA samples. This method is used in molecular biology. Briefly, purified DNA from a biological sample (such as blood or tissue) is digested with restriction enzymes, and the resulting DNA fragments are separated by electrophoresis using an electric current to move them through a sieve-like gel or matrix, which allows smaller fragments to move faster than larger fragments. The DNA fragments are transferred out of the gel or matrix onto a solid membrane, which is then exposed to a DNA probe labeled with a radioactive, fluorescent, or chemical tag. The tag allows any DNA fragments containing complementary sequences with the DNA probe sequence to be visualized within the Southern blot.

The Southern blotting combines the transfer of electrophoresis-separated DNA fragments to a filter membrane in a process called blotting, and the subsequent fragment detection by probe hybridization.

The method is named after the British biologist Edwin Southern, who first published it in 1975. 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 for compass directions as a sort of pun from Southern's name. As the label is eponymous, Southern is capitalized, as is conventional of proper nouns. The names for other blotting methods may follow this convention, by analogy.

History

Southern invented Southern blot after combining three innovations. The first one is the restriction endonucleases, which were developed at Johns Hopkins University by Tom Kelly and Hamilton Smith. Those restriction endonucleases are used to cut the DNA at a specific sequence. Kenneth and Noreen Murray introduced this technique as Southern. The second innovation is the gel electrophoresis that is based on separation of mixtures of DNA, RNA, or proteins according to molecular size, which was also developed at Johns Hopkins University, by Daniel Nathans and Kathleen Danna in 1971. The third innovation is the blotting-through method which was developed by Frederick Sanger, when he transferred RNA molecules to DEAE paper. Southern blot was invented in 1973 but it was not published until 1975. Although it was published later the technique was disseminated when Southern introduced the Southern blot technique to a scientist at Cold Spring Harbor Laboratory called Michael Mathews by drawing this technique on a paper.

Method

The genomic DNA is digested with either one or more than one restriction enzyme, then the DNA fragments are size-fractionated by gel electrophoresis. Before the DNA fragments are transferred to a solid membrane which is either nylon or nitrocellulose membrane they are first denatured by alkaline treatment.

  1. DNA Isolation: The DNA to be studied is isolated from various tissues. The most suitable source of DNA is known as blood tissue. However, it can be isolated from different tissues (hair, semen, saliva, etc.).
  2. DNA digestion: Restriction endonucleases are used to cut high-molecular-weight DNA strands into smaller fragments. This is done by adding the desired amount of DNA which can be changed according to the probe used and the intricacy of the DNA, with the restriction enzyme, enzyme buffer and purified water. Then everything is incubated at 37 °C overnight.
  3. Gel electrophoresis: 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 before 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.
  4. Denaturation: 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.
  5. Blotting: 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 constantly 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. Five methods can be used to transfer DNA fragments to the solid membrane and they are:
  6. Can be used to find similar sequences in other species or in the genome by decreasing the specificity of hybridization.
  7. Can be used in personal identification through fingerprinting, and in disease diagnosis.

Limitations

  1. Compared to other tests, southern blot is a complex technique that has multiple steps and these steps require equipment and reagents that are expensive.