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Diagnosis of Fragile X Syndrome by Southern Blot Hybridization Using a Chemiluminescent Probe: A Laboratory Protocol

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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.

Methods and 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.

Conclusion: 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.

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Authors and Affiliations

  1. Research and Development Department, Quest Diagnostics, 7600 Tyrone Ave., Van Nuys, California, 91405

    Bert Gold PhD & Daniela Radu MS

  2. Genetic Testing Center, Quest Diagnostics, Van Nuys, California

    Alla Balanko BS & Chih-Sheng Chiang PhD

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  1. Bert Gold PhD
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  2. Daniela Radu MS
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  3. Alla Balanko BS
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  4. Chih-Sheng Chiang PhD
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Correspondence to Bert Gold PhD.

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Gold, B., Radu, D., Balanko, A. et al. Diagnosis of Fragile X Syndrome by Southern Blot Hybridization Using a Chemiluminescent Probe: A Laboratory Protocol. Molecular Diagnosis 5, 169–178 (2000). https://doi.org/10.1007/BF03262073

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  • DOI: https://doi.org/10.1007/BF03262073

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