Southern blot is a technique that uses gel electrophoresis and probes to detect and determine the amount of DNA in a sample. The process involves treating the DNA with restriction enzymes, separating it with electrophoresis, transferring it onto a sheet, incubating it with a probe, and revealing the locations where the probe has bound to the DNA. It is useful for identifying DNA sequences of interest, determining the number of copies of a sequence, and genetic manipulation of microorganisms.
Southern blot is a technique used to detect DNA in a sample and determine the amount of DNA present. This procedure is named after Edwin Southern, a British biologist who pioneered the technique in the 1970s. Southern blot analysis uses gel electrophoresis to separate the DNA fragments in a sample and then uses probes to detect the DNA sequences of interest.
Southern blotting begins with a sample of DNA that has been extracted from cells. The DNA is treated with restriction enzymes which split the nucleic acid into fragments of various sizes. Next, the DNA is subjected to denaturing agarose gel electrophoresis. This process separates the fragments into individual strands while they are also separated by size.
The next step of the Southern blot is to transfer the separated DNA fragments onto a sheet of nylon or nitrocellulose, which keeps the fragments immobile. Subsequently, the sheet is incubated with a single-stranded DNA hybridization probe. The probe sequence is designed to bind to the DNA sequence that the experiment is trying to detect. Each probe fragment will bind to its complementary DNA sequence, if present in the sample, to form a double-stranded section of DNA. The probe is labeled with a radioactive isotope or 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 the locations where the probe has bound to the complementary DNA. If the probe has been tagged with a radioactive isotope, this is done by using X-rays to detect where the probe has hybridized to sample DNA fragments. When an enzyme is used, an additional incubation is performed. The enzyme used for this purpose is one that produces a colored product when it reacts with its substrate, so the Southern blot is completed by adding a substrate to reveal the locations where the probe DNA has bound to the 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 of interest or that need to 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 multiple copies of a sequence are present and bound to the probes.
Another common use of the technique is in the genetic manipulation of bacteria and other microorganisms. In this case the bacterial DNA is probed to determine if foreign DNA has been successfully introduced into the bacteria. Southern blotting is also the basis for the restriction fragment length polymorphism technique.
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