Southern blotting: detecting a specific DNA sequence
Southern blotting detects a specific DNA sequence in a sample, because a probe (a piece of DNA carrying a radioactive or fluorescent label) pairs with its matching sequence.
Remember
Denaturation separates the DNA into single strands so that the probe can pair with its bases, and the probe's own sequence then picks out the target; the other steps prepare the DNA or show where the probe ended up.
Memory aid
the three blots follow the compass, with Southern for DNA, Northern for RNA and Western for protein
Use the compass direction in the name of the blot to recall which molecule it detects.
A Southern blot picks out one DNA sequence from many fragments. The DNA is cut with restriction enzymes (proteins that cut at particular recognition sequences). Gel electrophoresis then sorts the fragments by size. The fragments are denatured (separated into single strands) and transferred onto a membrane, a support that holds the pattern in place.
Intact DNA is double stranded: its strands are held together by hydrogen bonds between the paired bases, so those bases are not free to pair with a probe. Denaturation separates the two strands, usually with an alkaline solution such as sodium hydroxide, and leaves single strands with their bases exposed. In the usual protocol this happens before transfer to the membrane.
The probe is a labelled piece of DNA whose sequence is complementary to the target, so it can pair with the bases of the target. Pairing between probe and target is called hybridisation. The probe stays attached where its sequence matches, so it marks the target fragment and not unrelated DNA. That is why the specificity comes from the probe and not from denaturation, electrophoresis or transfer.
Finally, the bound probe is detected, for example by autoradiography (recording the label on X-ray film) or by colour development. Detection reports where the probe has ended up, so it comes after hybridisation.
Key facts
- Purpose
- detects a specific DNA sequence within a DNA sample.
- Order of steps
- digest (cut with restriction enzymes), separate by size, denature, transfer to a membrane, add the probe, then detect.
- Why denature
- the probe pairs with bases that are free, and denaturation frees them by separating the two strands.
- Source of specificity
- hybridisation, where the probe pairs with its complementary sequence, rather than any of the other steps.
- Detection
- autoradiography or colour development shows where the probe has bound, so it follows hybridisation.
- Other blots
- Northern blotting detects RNA and Western blotting detects protein.
Exam traps
- Gel electrophoresis sorts fragments by size, which does not pick out a target sequence, because different genes can give fragments of similar size: it shows where fragments are, not which of them carries the target.
- Denaturation does not cut DNA into shorter pieces, because cutting is done by restriction enzymes and denaturation separates the strands.
- An antibody does not recognise the target DNA in a Southern blot, because antibodies are used in Western blotting to recognise proteins, while a labelled DNA probe pairs with the complementary sequence.
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