Invader chemistry at work.

AgBio's Invader® chemistry is composed of two simultaneous isothermal reactions. A primary reaction specifically and accurately detects single-base changes, insertions, deletions and changes in gene and chromosome number. A second reaction is used for signal amplification and generic readout.

In the first reaction, two oligonucleotides - a probe and an Invader oligo - hybridize to the target DNA region of interest to generate a one-base overlapping structure at the nucleotide being interrogated. If the sequence is present this triplex structure is recognized and cut at a specific site by our proprietary Cleavase® enzymes, resulting in release of the 5' arm (Flap) of the probe oligonucleotide (Figure 1A).

Figure 1: Primary Invader Reaction (Target Specific)

Figure 1A

Multiple 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 substrate is formed and cleavage occurs. The number of flaps released is proportional to the amount of target in the sample, allowing for quantitative detection of genes, chromosomes or infectious agents (Figure 1B).

Figure 1 (part 2): Primary Invader Reaction (Target Specific)

Figure 1B

This Flap then serves as the "Invader" oligo in the secondary reaction resulting in specific cleavage of a labeled universal oligonucleotide, the FRET Probe (Figure 2). Cleavage of this FRET probe results in the generation of a fluorescent signal. When the targeted sequence is not present in the sample tested, cleavage does not occur, the Flap is not released, and a fluorescent signal is not generated.

Figure 2

Utilizing different 5' Flap sequences and their corresponding FRET probes with non-overlapping fluorophores allows for multiple sequences to be detected in a single well. Comparing the signal generated from each dye in the Invader reaction will yield an objective genotype call.

AgBio's Invader assay can be performed with standard laboratory instrumentation, keeping equipment costs modest. The assay can be performed with manual 96-well set-up and is easily amenable to fully automated platforms with 384 well or higher density formats. The reaction volume is scalable to sub-microliter reaction volumes, while maintaining the assay's specificity and accuracy.