Southern Blotting and Related DNA Detection Techniques

Southern blotting is one of the central techniques in molecular biology. First devised by E.M. Southern (1975), Southern blotting results in transfer of DNA molecules, usually restriction fragments, from an electrophoresis gel to a nitrocellulose or nylon sheet (referred to as a ‘membrane’), in such a way that the DNA banding pattern present in the gel is reproduced on the membrane. During transfer or as a result of subsequent treatment, the DNA becomes immobilized on themembrane and can be used as a substrate for hybridization analysis with labelled DNA or RNA probes that specifically target individual restriction fragments in the blotted DNA. In essence, Southern blotting is therefore a method for ‘detection of a specific restriction fragment against a background of many other restriction fragments’ (Brown, 1999). The restricted DNA might be a plasmid or bacteriophage clone, Southern blotting being used to confirm the identity of a cloned fragment or to identify an interesting subfragment from within the cloned DNA, or it might be genomic DNA, in which case Southern blotting is a prelude to techniques such as restriction fragment length polymorphism (RFLP) analysis. In this article the general principles of Southern blotting are described, followed by overviews of the methodologies used for preparation ofDNAprior to blotting, for blotting itself, and for hybridization analysis. The article concludes with a survey of the applications and limitations of Southern blotting. The Principle of the Southern Blot