Master the Southern Blot Procedure

The Southern Blot Procedure: A Versatile Tool for DNA Fragment Detection

• The Southern blot procedure is used to detect specific DNA fragments.
• The procedure takes advantage of the fact that complementary polynucleotide strands can anneal to form double-stranded molecules.
• The technique was invented by Edwin M. Southern in 1975 and is named after him.
• The procedure uses nitrocellulose or nylon membranes to tightly bind single-stranded DNA fragments.
• One or more restriction endonucleases digest DNA, and the resulting fragments are separated according to size by gel electrophoresis.
• The gel is soaked in a sodium hydroxide solution to denature the DNA and transferred to a nitrocellulose or nylon membrane.
• The single-stranded DNA molecules bind to positions on the membrane identical to their positions on the agarose gel, preserving the band pattern.
• The membrane is then incubated with a complementary single-stranded DNA or RNA probe with a detectable label to identify DNA sequences of interest.
• The Southern blot procedure is a powerful tool that detects changes in DNA that alter restriction cutting sites or the lengths of segments between restriction cutting sites.
• It has become an important clinical laboratory tool for identifying genetic problems.
• Investigators usually prefer to use a DNA or RNA probe with a fluorescent tag when performing the Southern blot procedure.
• This allows them to see fluorescent bands of interest without performing the time-consuming autoradiography step and can work without taking the precautions needed when working with radioisotopes.