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. After the DNA fragments are immobilized on the membrane, prehybridization methods are used to reduce non-specific probe binding. Then the fragments on the membrane are hybridized with either radiolabeled or nonradioactive labeled DNA, RNA, or oligonucleotide probes that are complementary to the target DNA sequence. Then detection methods are used to visualize the target DNA.

Interpretation of results

Hybridization of the probe to a specific DNA fragment on the filter membrane indicates that this fragment contains a 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. Southern blots performed with restriction enzyme-digested genomic DNA may be used to determine the number of sequences (e.g., gene copies) in a genome. A probe that hybridizes only to a single DNA segment that has not been cut by the restriction enzyme will produce a single band on a Southern blot, whereas multiple bands will likely be observed when the probe hybridizes to several highly similar sequences (e.g., those that may be the result of sequence duplication). To improve specificity and reduce hybridization of the probe to sequences that are less than 100% identical, the hybridization parameters may be changed (for instance, by raising the hybridization temperature or lowering the salt content). Nylon membrane is more durable and has higher binding capacity to DNA fragments than nitrocellulose membrane, so the DNA fragments will be more fixed to the membrane even when the membrane is incubated in high temperatures. In addition, compared to the nitrocellulose membrane which requires a high ionic strength buffer to bind the DNA fragments to the membrane, nylon charged membranes use buffers with very low ionic strength to transfer even small fragments of DNA of about 50 bp to the membrane, usually the DNA to be transferred is separated by polyacrylamide gel. In the blotting step the most efficient method to transfer the DNA from the gel to the membrane is the vacuum transfer since it transfers the DNA more rapidly and quantitatively.

Applications

Limitations