Written and maintained by CASRAI Editorial Board
Last updated
The Southern blot detects a specific DNA sequence within a complex mixture — classically an entire genome — by separating restriction fragments on a gel, immobilising them on a membrane, and finding the fragment of interest with a labelled probe. Edwin Southern described it in 1975, in a paper titled Detection of specific sequences among DNA fragments separated by gel electrophoresis. Northern (RNA) and Western (protein) blotting are named in play on his surname.
It has been partly displaced by PCR and sequencing for routine detection, but it remains the reference method where you need to see the actual genomic context: copy number, insertion site, integration structure, and rearrangements that an amplification-based assay will happily miss because it only reports what its primers can reach.
The sequence, and why each step exists
Every step in a Southern blot solves a specific physical problem. Skipping one because the protocol is long is the most common reason blots fail.
- Restriction digest. Genomic DNA is cut with restriction enzymes into fragments of resolvable size. The choice of enzyme determines what you can conclude — a site inside your region of interest gives a different band pattern from one flanking it, and that difference is the whole experiment when you are mapping an insertion.
- Agarose gel electrophoresis. Fragments are separated by size.
- Depurination — typically 0.25 N HCl for 15 minutes at room temperature with shaking. Brief acid treatment nicks large fragments so they transfer out of the gel efficiently. Large DNA otherwise moves out of an agarose matrix very poorly, and high-molecular-weight bands come out faint or absent.
- Alkaline denaturation — 0.5 M NaOH with 1.5 M NaCl, 30–60 minutes at room temperature. The probe can only hybridise to single-stranded DNA, so the duplex must be separated before transfer.
- Neutralisation, unless you are running an alkaline transfer, which uses the denaturant as the transfer buffer.
- Capillary transfer upward from gel to a nylon or nitrocellulose membrane in a high-salt buffer. The salt is not incidental — it promotes DNA binding to the membrane.
- Immobilisation by UV crosslinking or baking, so the DNA stays put through hybridisation and repeated washes.
- Prehybridisation and hybridisation with a labelled probe.
- Stringency washes, then detection.
Stringency is the parameter that decides your answer
The washes are where a Southern blot is actually tuned, and they are the step most often treated as boilerplate.
Wash buffers run from 2× SSC (low stringency) down to 0.2× SSC (high stringency), with 0.2% SDS held constant throughout. Lower salt and higher temperature destabilise imperfect duplexes, so the more stringent the wash, the closer a match the probe must be to stay bound.
That gives you a deliberate choice rather than a fixed recipe:
- High stringency when you want only the exact target — confirming a single-copy insertion, for example.
- Low stringency when you are deliberately looking for related sequences: gene family members, homologues in another species, partially diverged repeats.
A blot showing more bands than you expected is therefore not automatically contaminated or wrong. It may be reporting real cross-hybridisation to related sequences, and the way to find out is to raise the stringency and see which bands survive rather than to repeat the whole blot.
Where Southern blotting still beats PCR
PCR answers “is this sequence present, and how much”. A Southern blot answers “in what genomic context”, which is a different question.
- Copy number and integration structure in transgenic lines — band count and size report how many insertions there are and whether they are intact.
- Large rearrangements, deletions and insertions that shift a restriction fragment size. An amplicon spanning only part of the locus can look entirely normal.
- Repeat expansions, which are notoriously difficult to amplify faithfully.
- Confirming an edit did what you think — a PCR product proves a sequence exists somewhere; a Southern shows the surrounding fragment.
Common failures and their causes
- Faint or missing high-molecular-weight bands — inadequate depurination, so large fragments never left the gel. Check that step before blaming the probe.
- No signal at all — incomplete denaturation (the probe cannot bind duplex DNA), failed crosslinking, or a probe that was not successfully labelled. Confirm the probe independently before rerunning the transfer.
- High background across the membrane — insufficient prehybridisation blocking, too much probe, or washes that were too gentle.
- Unexpected extra bands — genuine cross-hybridisation at low stringency, star activity or partial digestion in the restriction step. Rule out digestion first by inspecting the ethidium-stained gel.
- Smeared lanes — degraded genomic DNA, or overloading. Southern blotting needs intact high-molecular-weight input; it is unforgiving of DNA that has been vortexed or repeatedly frozen.
- Bubbles under the membrane — produce blank patches that read as absent signal. They are introduced at assembly and cannot be fixed afterwards.
Related methods
The digest that starts a Southern blot is ordinary restriction-enzyme digestion, and the separation step is standard agarose gel electrophoresis — if either is poor, no amount of care downstream recovers the blot. Where the question is the exact base sequence rather than fragment structure, Sanger sequencing answers it directly.
Frequently asked questions
What does a Southern blot actually detect?
A specific DNA sequence within a complex mixture, reported as a band whose size tells you which restriction fragment carries it. Both facts matter: presence, and genomic context.
Why is depurination necessary?
Large DNA fragments transfer out of agarose poorly. Brief acid treatment — 0.25 N HCl for about 15 minutes — nicks them so they move efficiently. Omit it and your largest bands are the ones you lose.
Why denature with NaOH before transfer?
Hybridisation requires single-stranded targets. Alkaline treatment in 0.5 M NaOH with 1.5 M NaCl separates the strands so the probe can bind after transfer.
What is the difference between Southern, Northern and Western blotting?
The target. Southern detects DNA, Northern detects RNA, Western detects protein. Only Southern is named after a person; the others are puns on it.
How do I choose wash stringency?
By what you are asking. High stringency (towards 0.2× SSC) isolates exact matches; low stringency (2× SSC) deliberately retains related sequences. Keep 0.2% SDS constant and vary salt and temperature.
Is Southern blotting obsolete?
No, though it is no longer routine. It remains the reference method for copy number, integration structure and large rearrangements — the questions where a PCR product can be perfectly normal and still be hiding the answer.
References
- Southern blotting — Nature Protocols
- Southern blotting: capillary transfer of DNA to membranes — Nature Methods
- Southern blotting — PubMed 18429082
- A dual-colour Southern blot to visualise two genomes or genic regions simultaneously — PMC4010645
