1. Rapid identification of female carriers of DMD/BMD by quantitative real-time PCR
- Author
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Barbara Neuhaus, Franziska Joncourt, Kristin Jostarndt-Foegen, Stephanie Kleinle, Bernhard Steiner, and Sabina Gallati
- Subjects
Time Factors ,Diamines ,Polymerase Chain Reaction ,Dystrophin ,Exon ,chemistry.chemical_compound ,Gene Duplication ,Gene duplication ,Genetics ,Humans ,Benzothiazoles ,Organic Chemicals ,Allele ,Genetics (clinical) ,Fluorescent Dyes ,Sequence Deletion ,biology ,Genetic Carrier Screening ,Reproducibility of Results ,Muscular Dystrophy, Duchenne ,Rapid identification ,Quantitative Real Time PCR ,chemistry ,Quinolines ,SYBR Green I ,Carrier status ,biology.protein ,Female - Abstract
Recently developed PCR systems offer online-monitoring of amplification and allow simple and reliable DNA quantification. We have used the LightCycler system to develop a simple and rapid method for direct identification of female carriers of deletions and duplications in the dystrophin gene. The challenge resides in the ability to identify the presence of a deleted or duplicated allele over the background contributed by the normal allele. Quantification is based on the determination of the ratio between potentially deleted/duplicated dystrophin exons and non-deleted/-duplicated reference exons using the unspecific dsDNA-dye SYBRgreen I. In a retrospective study, we evaluated our method in female relatives of DMD/BMD patients with known carrier status by comparative analysis of deleted or duplicated versus non-deleted/-duplicated exons. Carrier status was accurately attributed in 100% of cases, the mean ratios being 0.52±0.12 for deletion carriers (expected value: 0.5) and 1.56±0.18 for duplication carriers (expected value: 1.5) vs. 1.022±0.17 for non-carriers (expected value: 1.0). The method proved to be simple, rapid, reliable, and cost-effective. It may be used for direct determination of deletions/duplications in potential DMD/BMD carriers and may easily be adapted for other genetic conditions involving deletions and duplications. Hum Mutat 23:385-391, 2004 © 2004 Wiley-Liss, Inc.
- Published
- 2004