Primary Reaction

In the primary reaction, two oligonucleotides, a probe and an Invader^® oligo, anneal to a specific DNA target sequence to generate a one-base overlapping structure if the desired sequence is present. The one-base overlapping structure is created with the probe and the Invader oligo on the target. The proprietary Cleavase^® enzyme specifically cleaves the overlapping primary probe, releasing the 5' flap plus one nucleotide.

Probes cycle rapidly on and off the target during the primary reaction. Each time an intact probe molecule binds to the specific target in the presence of the Invader^® oligo, the overlapping invasive structure is formed and cleavage occurs. The number of flaps released is relative to the amount of target in the sample, allowing for quantitative detection of genes, chromosomes or infectious agents.

Secondary, Simultaneous Reaction

Cleaved flaps from the primary Invader^® reaction combine with a fluorescence resonance energy transfer (FRET) probe in a secondary, simultaneous cleavage reaction, generating a fluorescent signal. The combination of two different flap sequences, FRET oligos, and fluorophores allows for single-well biplex reactions to occur.