High-Speed Detection of the G894T Polymorphism in Exon 7 of the eNOS Gene by Real-Time Fluorescence PCR with the Light-Cycler |
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Authors: | Elena Sticchi Cinzia Fatini Francesca Gensini Beatrice Battaglini Agatina Alessandrello Liotta Rosanna Abbate |
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Institution: | Department of Clinical Physiopathology, Unit of Medical Genetics, University of Florence, Florence, Italy. |
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Abstract: | In endothelial cells nitric oxide (NO) is synthesized by endothelial-nitric oxide synthase (e-NOS), constitutively expressed and encoded by a 26-exon gene, located on chromosome 7q35-36. The prevalence of the T rare variant of the G894T polymorphism in exon 7 of the e-NOS gene (Glu-->Asp amino acid substitution) has been reported to be significantly higher in patients with coronary spasm and coronary artery disease. To date G894T polymorphism detection is performed by PCR-RFLP assay. In order to establish a high-speed genotyping method, we have taken advantage of the Light Cycler instrument, a thermal cycler that combines rapid-cycle DNA amplification with a real-time fluorescence monitoring. This technology is based on hybridization of the adjacent fluorescently labeled probes with PCR products. This methodology is considered more accurate and less time-consuming than conventional PCR-RFLP assay. To validate this technique we genotyped 270 healthy subjects. The results were consistent with those obtained from PCR-RFLP assay. |
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