Southern blotting is used to analyse DNA. A restriction enzyme digest of genomic DNA is electrophoresed, transferred to a nylon membrane, and hybridised with a labelled probe. In which clinical scenario is Southern blotting the gold standard diagnostic tool?

• A Detection of point mutations in CFTR (cystic fibrosis)
• B Sequencing of mitochondrial DNA mutations
• C Quantification of mRNA expression levels in tissues
• D Detection of CGG triplet repeat expansions >200 in the FMR1 gene (Fragile X syndrome) ✓

Explanation

Fragile X syndrome is caused by CGG trinucleotide repeat expansion in the 5' UTR of FMR1. When repeats exceed ~200 (full mutation), the region becomes methylated, silencing FMR1. PCR fails to amplify very large expanded alleles efficiently because of the high GC content and repetitive structure. Southern blotting detects: (1) the large size shift of the expanded allele (restriction fragment longer than normal), and (2) methylation status (using methylation-sensitive restriction enzymes). It reliably identifies full mutations in males and females and distinguishes premutations (55–200 repeats) from full mutations. PCR is used for premutations but Southern blot remains gold standard for full mutations.

Reference: Harper's Illustrated Biochemistry, 32nd ed.

High-yield for: NEET PGINI-CETNExTFMGEUSMLEPLABMRCP

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