This technique is usually used in molecular genetic diagnostics to assess large repeat expansions or contractions, such as those exceeding 110 CTG trinucleotide repeats, and to evaluate DNA methylation status. Genomic DNA is first digested with restriction enzymes that are sensitive to methylation, chosen based on the specific DNA sequence under analysis. The digested fragments are then separated by size through agarose gel electrophoresis and transferred to a nylon membrane as part of the Southern blot process.

The DNA on the membrane is hybridised with a labelled probe, which can be radioactive but is increasingly non-radioactive. In non-radioactive methods, detection involves an indirect process using a fluorescence-labelled antibody after exposure to an X-ray film placed over the membrane. The resulting band patterns on the developed film are analysed and interpreted.

Southern blot analysis is particularly useful in the molecular genetic examination of triplet repeat disorders, such as , where it allows for the sizing of large repeat expansions (>110 CTG) that cannot be measured through capillary electrophoresis-based fragment length analysis. It is also crucial in investigating conditions like , where it helps determine if triplet repeat expansions lead to methylation of the affected region, which, in turn, can inhibit transcription and result in the loss of the FMR1 gene product.

Although time-consuming, this method is a reliable and well-established tool in genetic diagnostics. In some cases, the use of different restriction enzymes and probes is required, but it generally provides only an approximate estimate of repeat numbers. Accurate interpretation of band patterns requires expert knowledge.