text: Flap endonuclease (FEN) is an endonuclease that catalyzes structure-specific cleavage.  This cleavage is highly sensitive to mismatches and can be used to interrogate SNPs with a high degree of specificity
  In the basic Invader assay, a FEN called cleavase is combined with two specific oligonucleotide probes, that together with the target DNA, can form a tripartite structure recognized by cleavase.  The first probe, called the Invader oligonucleotide is complementary to the 3’ end of the target DNA.  The last base of the Invader oligonucleotide is a non-matching base that overlaps the SNP nucleotide in the target DNA.  The second probe is an allele-specific probe which is complementary to the 5’ end of the target DNA, but also extends past the 3’ side of the SNP nucleotide.  The allele-specific probe will contain a base complementary to the SNP nucleotide. If the target DNA contains the desired allele, the Invader and allele-specific probes will bind to the target DNA forming the tripartite structure.  This structure is recognized by cleavase, which will cleave and release the 3’ end of the allele-specific probe. If the SNP nucleotide in the target DNA is not complementary to the allele-specific probe, the correct tripartite structure is not formed and no cleavage occurs.  The Invader assay is usually coupled with fluorescence resonance energy transfer (FRET) system to detect the cleavage event. In this setup, a quencher molecule is attached to the 3’ end and a fluorophore is attached to the 5’ end of the allele-specific probe.  If cleavage occurs, the fluorophore will be separated from the quencher molecule generating a detectable signal.
  Only minimal cleavage occurs with mismatched probes making the Invader assay highly specific.  However, in its original format, only one SNP allele could be interrogated per reaction sample and it required a large amount of target DNA to generate a detectable signal in a reasonable time frame. Several developments have extended the original Invader assay. By carrying out secondary FEN cleavage reactions, the Serial Invasive Signal Amplification Reaction (SISAR) allows both SNP alleles to be interrogated in a single reaction.  SISAR Invader assay also requires less target DNA, improving the sensitivity of the original Invader assay.  The assay has also been adapted in several ways for use in a high-throughput format.  In one platform, the allele-specific probes are anchored to microspheres. When cleavage by FEN generates a detectable fluorescent signal, the signal is measured using flow-cytometry. The sensitivity of flow-cytometry, eliminates the need for PCR amplification of the target DNA (Rao et al. 2003). These high-throughput platforms have not progressed beyond the proof-of-principle stage and so far the Invader system has not been used in any large scale SNP genotyping projects.