sybr-green-i and Muscular-Dystrophy--Duchenne

To develop a robust and reliable assay for direct identification of female carriers of deletions in the dystrophin gene.. We compared two quantitative real-time PCR approaches for the detection of the deletions of exons 4, 17, 47, and 50 in DMD/BMD carriers. One hundred and ten individuals from 26 unrelated families, including 8 large pedigrees characterized by having at least two DMD affected males, were studied. Carrier status of the subjects was also evaluated by MLPA.. The results showed the gene dosage ratio of 0.99+/-0.14 and 1.09+/-0.19 for normal individuals and 0.48+/-0.06 and 0.50+/-0.10 for carriers in SYBR green and TaqMan probe assays, respectively. Carrier status was accurately attributed in 100% of cases and confirmed by MLPA.. Quantitative real-time PCR can be used as a direct method for carrier detection in female relatives of DMD patients with known deletions. The results are comparable to the MLPA data.

Topics: Benzothiazoles; Diamines; DNA Mutational Analysis; Dystrophin; Exons; Female; Fluorescent Dyes; Gene Deletion; Heterozygote; Humans; Male; Muscular Dystrophy, Duchenne; Organic Chemicals; Pedigree; Polymerase Chain Reaction; Quinolines; Reproducibility of Results

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.

Topics: Benzothiazoles; Diamines; Dystrophin; Female; Fluorescent Dyes; Gene Duplication; Genetic Carrier Screening; Humans; Muscular Dystrophy, Duchenne; Organic Chemicals; Polymerase Chain Reaction; Quinolines; Reproducibility of Results; Sequence Deletion; Time Factors