Diagnosis of Fragile X syndrome by Southern blot hybridization using a chemiluminescent probe: A laboratory protocol

Background: Unequivocal molecular characterization of the FMR-I triplet exp ansion region requires the combined use of PCR to amplify normal- and premu tation-length alleles and Southern analysis to detect fully expanded allele s and assess methylation. We provide a detailed laboratory protocol, which can be generalized, for the preparation and use of a digoxigenin (DIG)-labe led probe for Southern analysis of genomic DNA digested with EcoR I and Eag I. Methods and Results: The StB12.3 probe cloned in a recombinant plasmid is l abeled by PCR amplification using M13 primers, in the presence of DIG-11-dU TP. Hybridization signal is visualized on x-ray film using an alkaline phos phatase anti-DIG-Fab conjugate in the presence of chemiluminescent substrat e CDP-Star (Tropix, Bedford, MA). We provide details of probe labeling and quantitation, preparation, and hybridization of the alkaline Southern blot and an analysis of data. Conclusion: Several publications describe PCR-based methods that claim to p reclude the requirement of Southern analysis for the diagnosis of Fragile X syndrome. However, none of these is as robust as the method described here . Currently, rapid Southern analysis is an important part of molecular dete ction of all possible normal and abnormal FMR-1 alleles. This nonradioactiv e approach is a convenient and rapid alternative to using a radioactive pro be.