Southern blotting

Southern blotting may be used to diagnose patients suspected of having conditions caused by large expansions of tandemly repeated DNA sequences (for example, those seen in myotonic dystrophy type 1). It can also provide information on the methylation status of DNA (for example, on the full expansions seen in fragile X syndrome patients).

Southern blotting is not routinely used within the NHS Genomic Medicine Service, having largely been replaced by less labour-intensive PCR-based alternatives. For more information, see short tandem repeat (STR) testing.

• Restriction enzymes are used to cut patients’ genomic DNA.
• The digested DNA is run on an agarose gel to size-separate fragments. While on the gel, the DNA is denatured to make it single-stranded.
• The size-separated, single-stranded DNA is transferred (‘blotted’) onto a membrane made of nylon or nitrocellulose.
• A DNA probe complementary to the genomic DNA sequence of interest is hybridised to the membrane.
• A washing step is used to remove any non-specifically-bound probes.
• The probe is labelled with a chemical or fluorescent tag, or historically with a radioactive tag. This is used to visualise the locations of the DNA fragments of interest on the membrane.
• The size of fragments of interest in the patient’s DNA sample can then be compared to a ‘ladder’ of fragments of known sizes and to control samples tested alongside the patient sample. The estimated size of the fragments seen in the patient is used to infer whether an expansion of a tandem repeat is present.

Where methylation detection is needed, the enzymes used in the first step can be selected to include those which are methylation-sensitive (that is, they cannot cut methylated DNA). Interpretation of the size pattern of the resulting fragments can provide clarity on whether the patient DNA is methylated.

Advantages

• Southern blotting does not rely on PCR amplification of DNA. This means that repeat expansions that are too large to amplify through PCR can be accurately sized.
• For some patients, it may be the only method that can provide accurate sizing of their repeat expansion. This can be important for conditions in which the severity of the phenotype depends on the size of the expansion (for example, myotonic dystrophy type 1).
• In fragile X syndrome, southern blotting can provide information on the methylation status of repeat expansions, which is not provided by alternative methods of STR testing. Fully expanded fragile X alleles detected through alternative methods are usually reported with the caveat that they are assumed to be methylated, because it isn’t actually tested.

Limitations

• A relatively large amount of input DNA is required.
• It is labour-intensive.
• It is not scalable (only a limited number of samples can be processed simultaneously).
• Many laboratories have discontinued their southern blotting services.
• Rarely, variants in restriction enzyme cutting sites can lead to atypical results.

For clinicians

• National Human Genome Research Institute: Southern blot