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Comparison of three methods for genotyping of prothrombotic polymorphisms |
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| Authors: | Marika Bianchi Enzo Emanuele Annalisa Davin Stella Gagliardi Emanuela Cova Valentina Meli Rosita Trotti Cristina Cereda |
| Affiliation: | 1. Laboratory of Neurogenetics, IRCCS Neurological Institute “C. Mondino”, Via Mondino 2, 27100, Pavia, Italy 2. Department of Health Sciences, University of Pavia, Via Bassi 21, 27100, Pavia, Italy 3. Laboratory of Experimental Neurobiology, IRCCS Neurological Institute “C. Mondino”, Via Mondino 2, 27100, Pavia, Italy 4. Laboratory of Clinical Biochemistry, IRCCS Neurological Institute “C. Mondino”, Pavia, Italy |
| Abstract: | Several methods have been developed to detect common prothrombotic mutations, including factor V Leiden (G1691), prothrombin G20210A, and methylenetetrahydrofolate reductase (MTHFR) C677C. In this study, we compared the accuracy of three different molecular techniques, i.e.: (1) restriction enzyme digestion (RFLP), (2) real time with hybridization probes and final melting curve (Fluorescence Resonance Energy Transfer, FRET), and (3) real time with hydrolysis probes (TaqMan®). Sequencing was used as the reference standard. Our data showed that RFLPs analysis for the detection of prothrombotic mutations, albeit easy-to-perform, had a limited reliability for assessing correct genotypes. FRET analysis displayed higher resolution than RFLPs. Additionally, FRET analysis was faster and less tedious than sequencing. |
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