Southern blot

A Southern blot is a method used in molecular biology for detection of a specific DNA sequence in DNA samples. Southern blotting combines transfer of electrophoresis-separated DNA fragments to a filter membrane and subsequent fragment detection by probe hybridization. A Southern blot is a method used in molecular biology for detection of a specific DNA sequence in DNA samples. Southern blotting combines transfer of electrophoresis-separated DNA fragments to a filter membrane and 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 in reference to Edwin Southern's name. As the label is eponymous, Southern is capitalised, as is conventional of proper nouns. The names for other blotting methods may follow this convention, by analogy. 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.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). Modification of the hybridization conditions (for example, increasing the hybridization temperature or decreasing salt concentration) may be used to increase specificity and decrease hybridization of the probe to sequences that are less than 100% similar. Southern blotting transfer may be used for homology-based cloning on the basis of amino acid sequence of the protein product of the target gene. Oligonucleotides are designed so that they are similar to the target sequence. The oligonucleotides are chemically synthesized, radiolabeled, and used to screen a DNA library, or other collections of cloned DNA fragments. Sequences that hybridize with the hybridization probe are further analysed, for example, to obtain the full length sequence of the targeted gene. Southern blotting can also be used to identify methylated sites in particular genes. Particularly useful are the restriction nucleases MspI and HpaII, both of which recognize and cleave within the same sequence. However, HpaII requires that a C within that site be methylated, whereas MspI cleaves only DNA unmethylated at that site. Therefore, any methylated sites within a sequence analyzed with a particular probe will be cleaved by the former, but not the latter, enzyme.