Fragile X Syndrome is associated with moderate to severe mental retardation and a folate-sensitive site band on chromosome Xq27.3. Fragile X Syndrome is the most common inherited cause of developmental and learning disabilities, affecting approximately 1 in 4,000 males. Since the discovery of the fragile X mental retardation (FMR1) gene responsible for the syndrome, molecular, rather than cytogenetic, diagnosis of Fragile X syndrome has become the gold standard. Numerous molecular diagnostic centres worldwide use PCR and Southern blotting to characterize the size of the CGG repeats within the gene, expansion of which has been shown to be associated with the vast majority of cases of Fragile X syndrome. Fragile X syndrome is a triplet repeat disorder caused by expansions of a CGG repeat in the FMR1 gene to more than 220 triplets (full mutation) that usually coincide with hypermethylation and transcriptional silencing. The disease phenotype results from deficiency or loss of FMR1 protein (FMRP) and occurs in both sexes. The underlying full mutations arise exclusively on transmission from a mother who carries a pre-mutation allele (60-200 CGGs). Unequivocal molecular characterization of the FMR-1 triplet expansion region requires the combined use of PCR to amplify normal and pre-mutation-length alleles and Southern analysis to detect fully expanded alleles.

The blot depicts 10 µg (lanes 2 & 3) and 5 µg (lanes 4 & 5) Fragile X carrier female and normal male DNA, respectively. The DNAs were double digested with EcoRI and NruI and run on a 1% agarose gel. The DNAs were then blotted onto Sure Blot® CHEMI™ nylon membrane. The blot was probed with the Fragile X CHEMI™ DNA Probe overnight and processed according to the Sure Blot® CHEMI™ Hybridization and Detection Kit instruction manual. Anti-digoxigenin antibody was applied for 30 minutes, followed by post-incubation washes and application of CSPD® Chemiluminescent Substrate.

Sure Blot^® CHEMI™ Hybridization and
Detection Kit
Qbiogene's Sure Blot^® CHEMI™ Hybridization & Detection Kit is designed to provide high quality, non-radioactive detection of digoxigenin-labeled DNA probes hybridized to Sure Blot^® Nylon Membrane. As an integrated hybridization and detection system, Sure Blot^® CHEMI™ is quality controled to produce high signal and low background. Comparable sensitivity to 32P is typically observed for single-copy gene detection. Sufficient reagents are provided to process 20 x 150cm blots.

Kit Components: Nylon membrane (3000 cm²), prehybridization and hybridization solutions, blocking reagent, anti-DIG-antibody, diethanolamine, CSPD chemiluminescent substrate

Fragile X CHEMI™ DNA Probe
(Digoxigenin Labeled)
The Fragile X Syndrome is identified by an ampiflication of the FMR1 (CGG)n repeat region. Qbiogene's Fragile X CHEMI™ probe is a digoxigenin-labeled DNA probe (pFxa1NHE) which can be used in conjunction with the Sure Blot^® CHEMI™ Kit (Cat. No. S4250-KIT) to detect (CGG) repeats in the FMR-1 gene. The same results are obtained as when using the standard P32-labeled probes, but without radioactivity. The amplification of the FMR-1 gene is detected on Southern blots by an increase in the band sizes. Also provided is a ready-to-use mixture of unlabeled and digoxigenin-labeled Lambda DNA/Hind III Molecular Weight Markers for convenient visualization on both ethidium bromide-stained agarose gels and Southern blots.

Sufficient probe is provided to process at least 1500 cm², volume 250 µl. A HindIII digested Lambda DNA is added as
Molecular Weight Marker. Each vial contains a mixture of
labeled and unlabeled marker. The unlabeled marker can be visualized on agarose gel, the labeled marker serves as a digoxigenin- positive control on the blot.