What is Southern blotting
Southern blotting is one of the central techniques in molecular biology. This technique was first devised by E. M. Southern in 1975, resulting in transfer of DNA molecules from an electrophoresis gel to a nitrocellulose or nylon sheet referred to as a membrane. By this technique, 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 the membrane 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 a method to detect a specific restriction fragment against a background of many other restriction fragments. The restricted DNA might be a plasmid or bacteriophage clone, and Southern blotting is used to confirm the identity of a cloned fragment or to identify an interesting subfragment from within the cloned DNA. In many cases, Southern blotting is a technique used prior to techniques such as restriction fragment length polymorphism (RFLP) analysis.
The Principle of the Southern Blot
This technique is based on fact that nitrocellulose powder or sheets are able to bind DNA. This ability of nitrocellulose powder or sheets has been known for many years and was utilized in the 1950s and 1960s in various types of nucleic acid hybridization studies. In these early techniques the immobilized DNA was unfractionated, simply consisting of total DNA that was bound to nitrocellulose powder or spotted onto a nitrocellulose sheet. Later in the early 1970s, the introduction of gel electrophoresis methods that enable restriction fragments of DNA to be separated on the basis of their size boosted the development of techniques for the transfer of separated fragments from gel to nitrocellulose support. The procedure involving capillary transfer of DNA from the gel to a nitrocellulose sheet placed on top of it is simple and effective, and has now become routine method used in many molecular biology laboratories.
In a word, Southern blotting is a technique that enables a specific restriction fragment to be detected against a background of many other restriction fragments. The basic methodology for Southern blotting has not changed since the original technique was described in 1975, but modifications have been introduced many times across years for purpose to speed up the process and achieve a more efficient transfer. Southern blotting has many applications in molecular biology, including the identification of one or more restriction fragments that contain a gene or other DNA sequence of interest, and in the detection of RFLPs used in construction of genomic maps.
