A Southern blot is a molecular biology method for detecting specific DNA sequences in a complex sample by transferring DNA fragments from an electrophoresis gel to a membrane and hybridising them with a complementary labelled probe.
Explanation
The technique, named after its inventor Edwin Southern, begins with isolation of genomic DNA and digestion with restriction enzymes to generate fragments. The fragments are separated by size through agarose gel electrophoresis and then denatured in situ to produce single‑stranded DNA. The gel is placed in contact with a nitrocellulose or nylon membrane and, using capillary action, vacuum or electrotransfer, the DNA migrates out of the gel onto the membrane, where it becomes immobilised in the same spatial arrangement. After blocking nonspecific binding sites, a labelled single‑stranded nucleic acid probe complementary to the target sequence is incubated with the membrane. Probes may be radiolabelled, fluorescent or chemiluminescent. Any membrane‑bound fragments containing sequences that are complementary to the probe will hybridise to it. Unbound probe is washed away and the bound probe is detected by autoradiography or imaging systems. Southern blotting requires high‑quality DNA and careful handling but provides information about the size of target fragments and copy number that cannot easily be obtained by polymerase chain reaction alone.
Applications and examples
In medical genetics, Southern blots have been used to identify deletions in the dystrophin gene in patients with Duchenne muscular dystrophy and to characterise fragile X expansions. They were essential in early AIDS research for demonstrating integration of HIV proviral DNA into host genomes. Southern blotting is still used to confirm the insertion site and copy number of transgenes in engineered plants and animals and to verify complete gene knockouts in stem cell lines. Forensic laboratories once relied on restriction fragment length polymorphism profiling by Southern blotting to compare DNA patterns between individuals. Studies of hepatitis B virus and human papillomavirus integration into host DNA have also used this method. Although polymerase chain reaction and sequencing have largely supplanted Southern blotting for routine diagnostics, it remains a valuable tool when information about fragment size and genomic organisation is required.
Southern blotting laid the foundation for later nucleic acid blotting techniques and continues to provide a means of physically visualising specific DNA sequences within complex genomes.
Related Terms: DNA hybridization, Restriction fragment length polymorphism, Probe, Nucleic acid, PCR.