title: Comparison of three methods for genotyping of prothrombotic polymorphisms.
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.
authors.affiliation: Laboratory of Neurogenetics, IRCCS Neurological Institute "C. Mondino", Via Mondino 2, 27100, Pavia, Italy. marika.bianchi@gmail.com
authors.forename: Marika
authors.initials: M
authors.lastname: Bianchi
authors.forename: Enzo
authors.initials: E
authors.lastname: Emanuele
authors.forename: Annalisa
authors.initials: A
authors.lastname: Davin
authors.forename: Stella
authors.initials: S
authors.lastname: Gagliardi
authors.forename: Emanuela
authors.initials: E
authors.lastname: Cova
authors.forename: Valentina
authors.initials: V
authors.lastname: Meli
authors.forename: Rosita
authors.initials: R
authors.lastname: Trotti
authors.forename: Cristina
authors.initials: C
authors.lastname: Cereda
mesh_terms: D000328:Adult; D005260:Female; D031541:Fluorescence Resonance Energy Transfer; D005820:Genetic Testing / Q000379:methods*; D006801:Humans; D008297:Male; D009693:Nucleic Acid Hybridization / Q000379:methods; D016133:Polymerase Chain Reaction / Q000379:methods; D011110:Polymorphism, Genetic; D012150:Polymorphism, Restriction Fragment Length; D011516:Prothrombin / Q000235:genetics*; D017422:Sequence Analysis, DNA
chemical_list: D011516:Prothrombin