Real-time fluorescence genotyping of factor V Leiden during rapid-cycle PCR

A single-step method for factor V Leiden genotyping is presented that uses rapid-cycle PCR and simultaneous fluorescence analysis with resonance energy transfer probes. A fragment of the factor V gene containing the mutation is amplified asymmetrically through use of a primer labeled with Cy5 in the presence of a 3'-fluorescein-labeled probe that covers the mutation site. When the fluorescein probe is annealed to the extension product of the Cy5-labeled primer, the fluorophores are brought into close enough contact for resonance energy transfer to occur. As the temperature increases, the probe melts from its target, decreasing the resonance energy transfer. When the probe is complementary to the product strand, it melts at 65 degrees C; if the single-base mutation is present, the probe melts at 57 degrees C. Concurrent amplification and analysis from genomic DNA takes 20-45 min and requires no sample manipulation after the fluorescence thermal cycler is loaded.