Duchenne/Becker muscular dystrophy carrier detection using quantitative PCR and fluorescence-based strategies
| Autor: | M. Sartore, Eric F. Rappaport, T Parrella, Mayrand Pe, James M. Robertson, Elaine S. Mansfield, Saul Surrey, Lucero My, Paolo Fortina, Roger V. Lebo |
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| Jazyk: | angličtina |
| Rok vydání: | 1993 |
| Předmět: |
Male
Duchenne muscular dystrophy Biology Polymerase Chain Reaction Muscular Dystrophies law.invention chemistry.chemical_compound law Primer dimer Multiplex polymerase chain reaction medicine Humans Genetics (clinical) Polymerase chain reaction Fluorescent Dyes Gel electrophoresis Staining and Labeling Genetic Carrier Screening Reproducibility of Results medicine.disease Fluoresceins Molecular biology Thiazoles Real-time polymerase chain reaction chemistry Female Ethidium bromide Deoxyuracil Nucleotides Fluorescent tag |
| Popis: | Dystrophin gene deletions account for up to 68% of all Duchenne (DMD) and Becker (BMD) muscular dystrophy mutations. In affected males, these deletions can be detected easily using multiplex PCR tests which monitor for exon presence. In addition, quantitative dosage screening can discriminate female carriers. We previously analyzed multiplex PCR products by gel electrophoresis and quantitation of fluorescently labeled primers with the Gene Scanner™ in order to test carrier status. These 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 staining, which can be used to quantify the products of multiplex PCR. The first method uses the DNA intercalating thiazole orange dye TOTO-1 to stain PCR products after 20 cycles. In the second method, fluorescein-12,2′-dUTP is incorporated into products during PCR as a fluorescent tag for subsequent quantitative dosage studies. Both methods label all multiplexed exons including the 506 bp exon 48 fragment that is difficult to detect and quantify by standard ethidium bromide staining. Using this approach, we determined DMD/BMD carrier status in 24 unrelated families using a fluorescent fragment analyzer. Analysis of fluorochrome-labeled PCR products facilitates quantitative multiplex PCR for gene-dosage analysis. © 1993 Wiley-Liss, Inc. |
| Databáze: | OpenAIRE |
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