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

Abstract

The goal of this multicenter study was to evaluate the second-generation Invader 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 (PCR-RFLP) or allele-specific PCR (AS-PCR) analysis. The Invader assay is a PCR-independent methodology that uses a microtiter plate format. In the assay, a specific upstream Invader oligonucleotide and a downstream probe hybridize in tandem to a complementary DNA template and form a partially overlapping 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 oligonucleotide to direct cleavage of a fluorescence resonance energy transfer (FRET) probe in a second invasive cleavage reaction. Cleavage of this FRET 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 operator error and one from unknown causes), but were concordant on retesting. These results indicate that a simple microtiter plate-based Invader assay can reliably genotype clinical patient samples for the factor V (Leiden) point mutation directly from genomic DNA without prior target amplification.

Figure 1.

Schematic outline of the Invader assay for FVL, showing discrimination of single basepair changes by the Invader assay. The key to single-base discrimination for Invader technology depends both on a match between the WT or Mut probe and the target sequence at the base to be discriminated and an overlap of at least one base by the Invader oligonucleotide. The specific sequence in the target molecule (sample DNA) determines whether cleavage and subsequent fluorescent signal generation will occur. The structures formed in A are substrates recognized by the Cleavase enzyme, whereas the structure shown in B is a structure not recognized by the Cleavase enzyme. The specific base shown in the target sequence is the base to be discriminated.

Figure 2.

Distribution of ratios from the Invader assay results at the study sites for wild-type, heterozygous, and mutant genotypes, as determined by PCR-RFLP or AS-PCR. Note that the scale for the ordinate is logarithmic.