A multi-site study for detection of the factor V (Leiden) mutation from genomic DNA using a homogeneous invader microtiter plate fluorescence resonance energy transfer (FRET) assay

The goal of this multicenter study was to evaluate the second-generation In vader technology for detecting the factor V (Leiden) mutation directly from genomic DNA of different sample types. Invader assay results were compared with polymerase chain reaction-restriction fragment length polymorphism (P CR-RFLP) or allele-specific PCR (AS-PCR) analysis. The Invader assay is a P CR-independent methodology that uses a microtiter plate format. In the assa y, a specific upstream Invader oligonucleotide and a downstream probe hybri dize in tandem to a complementary DNA template and form a partially overlap ping structure. The Cleavase VIII enzyme recognizes and cuts this structure to release the 5' flap of the probe. This flap then serves as an Invader o ligonucleotide to direct cleavage of a fluorescence resonance energy transf er (FRET) probe in a second invasive cleavage reaction. Cleavage of this FR ET probe results in the generation of a fluorescent signal. The results of the Invader assay were 99.5% concordant with the PCR-based methods. Of the 372 samples tested once, only two gave discordant results (one from operato r error and one from unknown causes), but were concordant on retesting. The se results indicate that a simple microtiter plate-based Invader assay can reliably genotype clinical patient samples for the fatter V (Leiden) point mutation directly from genomic DNA without prior target amplification.