The Southern blot is a technique which is used to detect DNA in a sample, and determine how much DNA is present. This procedure is named for Edwin Southern, a British biologist who pioneered the technique in the 1970s. Southern blot analysis uses gel electrophoresis to separate DNA fragments in a sample and then uses probes to detect DNA sequences of interest.
Southern blotting starts with a sample of DNA which has been extracted from cells. The DNA is treated with restriction enzymes that cleave the nucleic acid into fragments of varying sizes. Next, the DNA is run through a denaturing agarose gel electrophoresis. This process separates the fragments into single strands at the same time as they are also separated by size.
The next step in the Southern blot is to transfer the separated DNA fragments to a sheet of nylon or nitrocellulose, which holds the fragments immobile. Following this the sheet is incubated with a hybridization probe of single-stranded DNA. The sequence of the probe is designed so that it will bind to the DNA sequence that the experiment is attempting to detect. Each probe fragment will bind to its complementary DNA sequence if it exists in the sample, to form a section of double-stranded DNA. The probe is labeled with a radioactive isotope, or with an enzyme. Multiple probes can be used in the same experiment.
When the incubation is complete, the final step in the Southern blot process is to reveal locations where the probe has bound to complementary DNA. If the probe was labeled with a radioactive isotope, this is done by using x-rays to detect spots where the probe has hybridized to sample DNA fragments. When an enzyme is used, a further incubation is carried out. The enzyme used for this purpose is one which produces a colored product when it reacts with its substrate, so the Southern blot is completed by adding substrate to reveal locations where probe DNA has bound to sample DNA.
Southern blots are useful for a variety of reasons. The simplest use of the technique is to use a probe to identify DNA sequences which are of interest, or which must be isolated for other uses. Southern blotting is also used to determine how many copies of a particular sequence are present in a sample. This is possible because a stronger radioactive or enzymatic signal is produced when more copies of a sequence are present and bound to probes.
Another common use of the technique is in genetic manipulation of bacteria and other microorganisms. In this case bacterial DNA is probed to determine if foreign DNA has successfully been introduced into the bacteria. Southern blotting is also the basis for the restriction fragment length polymorphism technique.
