title: Combined factor V leiden (G1691A) and prothrombin (G20210A) genotyping by multiplex real-time polymerase chain reaction using fluorescent resonance energy transfer hybridization probes on the Rotor-Gene 2000.
abstract: Several methods have been developed to detect common single point mutations in the factor V and prothrobin genes that are risk factors for thrombophilia. Most are based on PCR followed by restriction enzyme digestion and electrophoresis (RFLP), but gel analysis has certain limitations, and alternative detection methods, including real-time PCR, have therefore been developed. In this study we developed and evaluated a combined factor V Leiden and prothrombin (G20210A) genotyping method based on multiplex real-time PCR with fluorescent resonance energy transfer (FRET) hybridization probes on the Rotor-Gene 2000. Two hundred subjects were screened for the two mutations. The FRET assay clearly discriminated among wild-type, homozygous and heterozygous status for the two mutations, and the results were in full agreement with those of the RFLP assay. This robust FRET probe-based assay also has a higher throughput capacity than conventional methods, handling up to 72 samples in 90 min.
authors.affiliation: Service d'Hématologie Biologique et d'lmmunologie, Hôpital Louis Mourier, AP-HP, Colombes, France.
authors.forename: Nejma
authors.initials: N
authors.lastname: Ameziane
authors.forename: Maryse
authors.initials: M
authors.lastname: Lamotte
authors.forename: Jérôme
authors.initials: J
authors.lastname: Lamoril
authors.forename: Dominique
authors.initials: D
authors.lastname: Lebret
authors.forename: Jean-Charles
authors.initials: JC
authors.lastname: Deybach
authors.forename: Thomas
authors.initials: T
authors.lastname: Kaiser
authors.forename: Dominique
authors.initials: D
authors.lastname: de Prost
mesh_terms: D004247:DNA / Q000737:chemistry / Q000235:genetics; D004252:DNA Mutational Analysis / Q000379:methods; D015342:DNA Probes / Q000737:chemistry / Q000235:genetics; D005165:Factor V / Q000235:genetics*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005838:Genotype; D006579:Heterozygote; D006720:Homozygote; D006801:Humans; D017354:Point Mutation; D012150:Polymorphism, Restriction Fragment Length; D011516:Prothrombin / Q000235:genetics*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013696:Temperature
chemical_list: D015342:DNA Probes; C095381:factor V Leiden; D005165:Factor V; D011516:Prothrombin; D004247:DNA