High-speed detection of the G894T polymorphism in exon 7 of the eNOS gene by real-time fluorescence PCR with the Light-Cycler.
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.
Sticchi
Elena
E
Department of Clinical Physiopathology, Unit of Medical Genetics, University of Florence, Florence, Italy.
Fatini
Cinzia
C
Gensini
Francesca
F
Battaglini
Beatrice
B
Liotta
Agatina Alessandrello
AA
Abbate
Rosanna
R
D000483:Alleles; D019943:Amino Acid Substitution; D001224:Aspartic Acid / Q000378:metabolism; D004730:Endothelium, Vascular / Q000201:enzymology*; D005091:Exons; D005453:Fluorescence; D005787:Gene Frequency; D005820:Genetic Testing / Q000379:methods*; D006801:Humans; D019001:Nitric Oxide Synthase / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D015203:Reproducibility of Results; D013997:Time Factors
D001224:Aspartic Acid; D019001:Nitric Oxide Synthase