title: Diagnosis of Fragile X syndrome by Southern blot hybridization using a chemiluminescent probe: a laboratory protocol.
abstract: BACKGROUND
  Unequivocal molecular characterization of the FMR-1 triplet expansion region requires the combined use of PCR to amplify normal- and premutation-length alleles and Southern analysis to detect fully expanded alleles and assess methylation. We provide a detailed laboratory protocol, which can be generalized, for the preparation and use of a digoxigenin (DIG)-labeled probe for Southern analysis of genomic DNA digested with EcoR I and Eag I.
  
  
  RESULTS
  The StB12.3 probe cloned in a recombinant plasmid is labeled by PCR amplification using M13 primers, in the presence of DIG-11-dUTP. Hybridization signal is visualized on x-ray film using an alkaline phosphatase anti-DIG-Fab conjugate in the presence of chemiluminescent substrate 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.
  
  
  CONCLUSIONS
  Several publications describe PCR-based methods that claim to preclude 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 detection of all possible normal and abnormal FMR-1 alleles. This nonradioactive approach is a convenient and rapid alternative to using a radioactive probe.
authors.affiliation: Research and Development Department, Quest Diagnostics, Van Nuys, CA 91405, USA. bert.m.gold@questdiagnostics.com
authors.forename: B
authors.initials: B
authors.lastname: Gold
authors.forename: D
authors.initials: D
authors.lastname: Radu
authors.forename: A
authors.initials: A
authors.lastname: Balanko
authors.forename: C S
authors.initials: CS
authors.lastname: Chiang
mesh_terms: D015139:Blotting, Southern / Q000379:methods*; D004252:DNA Mutational Analysis; D004076:Digoxigenin / Q000494:pharmacology; D051860:Fragile X Messenger Ribonucleoprotein 1; D005600:Fragile X Syndrome / Q000175:diagnosis* / Q000235:genetics; D006801:Humans; D008163:Luminescent Measurements; D008297:Male; D009419:Nerve Tissue Proteins / Q000235:genetics; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction; D016601:RNA-Binding Proteins; D019680:Trinucleotide Repeat Expansion
chemical_list: D004076:Digoxigenin; D051860:Fragile X Messenger Ribonucleoprotein 1; D009419:Nerve Tissue Proteins; D015345:Oligonucleotide Probes; D016601:RNA-Binding Proteins; C497683:FMR1 protein, human