B Gold1, D Radu, A Balanko, C S Chiang. 1. Research and Development Department, Quest Diagnostics, Van Nuys, CA 91405, USA. bert.m.gold@questdiagnostics.com
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.
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.
Authors: Jill M Haenfler; Geena Skariah; Caitlin M Rodriguez; Andre Monteiro da Rocha; Jack M Parent; Gary D Smith; Peter K Todd Journal: Front Mol Neurosci Date: 2018-08-15 Impact factor: 6.261