Non-PCR-dependent detection of the factor V Leiden mutation from genomic DNA using a homogeneous invader microtiter plate assay.

BACKGROUND: The goal of this study was to compare the Invader technology for the direct detection of the Factor V Leiden mutation from genomic DNA with a conventional polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay. METHODS AND RESULTS: In the Invader assay, a specific upstream "invading" oligonucleotide and a partially overlapping downstream probe together form a specific structure when bound to complementary DNA template. This structure is recognized and cut at a specific site by the Cleavase enzyme, resulting in release of the 59 flap of the probe oligonucleotide. This fragment now serves as the "Invader" oligonucleotide with respect to synthetic secondary targets and secondary fluorescently labeled signal probes contained in the reaction mixture, resulting in specific cleavage of the secondary signal probe by the Cleavase enzyme. Fluorescence signal is generated when this secondary probe, labeled with dye molecules capable of fluorescence resonance energy transfer, is cleaved. Genomic DNAs isolated from peripheral blood buffy coats of patients previously tested for the Factor V Leiden mutation by PCR-RFLP were tested using an Invader assay specific for this mutation. In all 48 samples containing sufficient DNA for testing (30 normal, 16 heterozygous, 2 homozygous mutant), the genotype determined by the Invader assay was concordant with the PCR-RFLP results. CONCLUSION: These results indicate that a simple microtiter plate-based Invader assay can reliably genotype routine clinical patient samples for the Factor V Leiden point mutation without the need for PCR amplification, restriction enzyme digestion, or gel electrophoresis.