Comparison of three methods for genotyping of prothrombotic polymorphisms.
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.
Bianchi
Marika
M
Laboratory of Neurogenetics, IRCCS Neurological Institute "C. Mondino", Via Mondino 2, 27100, Pavia, Italy. marika.bianchi@gmail.com
Emanuele
Enzo
E
Davin
Annalisa
A
Gagliardi
Stella
S
Cova
Emanuela
E
Meli
Valentina
V
Trotti
Rosita
R
Cereda
Cristina
C
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
D011516:Prothrombin