Development of FRET Real time PCR method for the simultaneous detection of factor V Leiden (G1691A) and G20210A prothrombin mutations

چکیده مقاله

Factor V Leiden (G1691A) and prothrombin gene mutation are the most important risk factors for thrombosis.
The aim of the current investigation was to develop and organized technique based on Real time PCR with fluorescence resonance energy transfer (FRET) detection and melting curve analysis for the simultaneous detection factor V Leiden (FVL) and G20210A prothrombin mutations.
Specific primers and probes were utilized to amplify the appropriate segment of the factor V Leiden and prothrombin gene and fluorescence resonance energy transfer hybridization probes were utilized for discerment in a Rotor Gene 6000.
Melting temperature (Tm values) for the wild type FVL(G1691A) was at 62.5 .C ,whereas Tm values for mutant allele was at 67 . Tm values for the wild type prothrombin (G20210A) was at 50 , while melting temperature (Tm) for mutent genotype was at 60 .
Our findings showed that designed primers and probes in this study, are enable to identify these mutations with high sensitivity and complete specificity

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