DUCHENNE-BECKER MUSCULAR-DYSTROPHY CARRIER DETECTION USING QUANTITATIVE PCR AND FLUORESCENCE-BASED STRATEGIES

Dystrophin gene deletions account for up to 68% of all Duchenne (DMD) and Becker (BMD) muscular dystrophy mutations. In affected males, thes e deletions can be detected easily using multiplex PCR tests which mon itor for exon presence. In addition, quantitative dosage screening can discriminate female carriers. We previously analyzed multiplex PCR pr oducts by gel electrophoresis and quantitation of fluorescently labele d primers with the Gene Scanner(TM) in order to test carrier status. T hese multiplex PCR protocols detect DMD gene deletions adequately, but require up to 18 pairs of fluorochrome-labeled primers. We previously described two alternative fluorescent labeling strategies, each with approximately 1,000-fold greater sensitivity than ethidium bromide sta ining, which can be used to quantify the products of multiplex PCR. Th e first method uses the DNA intercalating thiazole orange dye TOTO-1 t o stain PCR products after 20 cycles. In the second method, fluorescei n-12,2'-dUTP is incorporated into products during PCR as a fluorescent tag for subsequent quantitative dosage studies. Both methods label al l multiplexed exons including the 506 bp exon 48 fragment that is diff icult to detect and quantify by standard ethidium bromide staining. Us ing this approach, we determined DMD/BMD carrier status in 24 unrelate d families using a fluorescent fragment analyzer. Analysis of fluoroch rome-labeled PCR products facilitates quantitative multiplex PCR for g ene-dosage analysis. (C) 1993 Wiley-Liss, Inc.