Before the DNA fragments are transferred to a solid membrane which is either nylon or nitrocellulose membrane it is first denatured by alkaline treatment. The genomic DNA is digested with either one or more than one restriction enzyme, then the DNA fragments are size-fractionated by gel electrophoresis. 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. Southern blot was invented in 1973 but it was not published until 1975. The third innovation is blotting-through methods which was developed by Frederick Sanger, when he transferred RNA molecules to DEAE paper. The second innovation is the gel electrophoresis that 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. Kenneth and Noreen Murray introduced this technique as Southern. 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. The names for other blotting methods may follow this convention, by analogy. As the label is eponymous, Southern is capitalized, as is conventional of proper nouns. 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 about Edwin Southern's name. The method is named after the British biologist Edwin Southern, who first published it in 1975. 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 tag allows any DNA fragments containing complementary sequences with the DNA probe sequence to be visualized within the Southern blot. 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. 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 using an electric current to move them through a sieve-like gel or matrix, which allows smaller fragments to move faster than larger fragments. This method is used in molecular biology. Thus the reverse procedure, though originally uncommon, enabled the one-at-a-time study of gene expression using northern analysis to evolve into gene expression profiling, in which many (possibly all) of the genes in an organism may have their expression monitored.Southern blot is a method used for detection and quantification of a specific DNA sequence in DNA samples. The use of DNA microarrays that have come into widespread use in the late 1990s and early 2000s is more akin to the reverse procedure, in that they involve the use of isolated DNA fragments affixed to a substrate, and hybridization with a probe made from cellular RNA.
In this procedure, the substrate nucleic acid (that is affixed to the membrane) is a collection of isolated DNA fragments, and the probe is RNA extracted from a tissue and radioactively labelled.
For smaller fragments denaturing polyacrylamide urea gels are employed.Īs in the Southern blot, the hybridization probe may be made from DNA or RNA.Ī variant of the procedure known as the reverse northern blot was occasionally (although, infrequently) used. A notable difference in the procedure in case of agarose gels, (as compared with the Southern blot) is the addition of formaldehyde which acts as a denaturant. The gels may be run on either agarose or denaturing polyacrylamide gels depending on the size of the RNA to be detected. This technique was developed in 1977 by James Alwine, David Kemp, and George Stark at Stanford University. It takes its name from the similarity of the procedure to the Southern blot procedure, named for biologist Edwin Southern, used to study DNA, with the key difference that, in the northern blot, RNA, rather than DNA, is the substance being analyzed by electrophoresis and detection with a hybridization probe. The northern blot is a technique used in molecular biology research to study gene expression.
0 kommentar(er)
