Not only is the Invader® chemistry revolutionary in form, but its outstanding speed in practice can save hours in the lab and significantly enhance throughput and cost-efficiency.
The 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 for genetic, pharmacogenetic and infectious diseases. A second reaction is used for signal amplification and generic readout.
In the first reaction, two oligonucleotides—a probe and an Invader® oligo—associated with a specific region of the target DNA to generate a one-base overlapping structure at the nucleotide being interrogated.
If the desired sequence is present, an overlapping structure is created with the probe and the Invader® oligo on the target. Proprietary Cleavase® enzymes specifically cleave the primary probes that form overlapping structures with the Invader® oligo, releasing the 5' flaps plus one nucleotide.
In the primary reaction, multiple probe molecules are cleaved per target molecule, and the signal generated from the cleaved 5' flap is amplified. The probes cycle rapidly on and off the target; 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 can occur. 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.
Released flaps from the primary reaction serve as Invader® oligos in a second, simultaneous overlapping cleavage reaction on a labeled, synthetic oligo, the fluorescence resonance energy transfer (FRET) probe. Cleavage of this FRET probe results in the generation of a fluorescent signal. Using two different 5' flap sequences and their complementary FRET oligos with nonoverlapping fluorophores allows for two distinct sequences to be detected in a single well.
Each released 5' flap from the primary reaction cycles on and off the cleaved and uncleaved FRET probes, enabling cleavage of many FRET probes in the secondary reaction to further amplify the target-specific signal.
Because these two cleavage reactions occur simultaneously, they can produce 1 million to 10 million labeled cleavage products per target sequence. A standard 4-hour reaction produces more than 10 million-fold signals.
A simple, scalable, robust technology for any size CLIA high-complexity laboratory seeking to develop assays that increase throughput, lower costs and improve turnaround time.
The single-tube reaction setup, objective analysis, automation readiness, and flexibility of the Invader® chemistry can enable numerous applications, including:
Third Wave Technologies offers an In Vitro Diagnostic (UGT1A1) and Invader® analyte-specific reagents* (ASRs) for mutations associated with:
*Analyte Specific Reagent. Analytical and performance characteristics are not established.
The Invader® chemistry is protected by foreign and domestic patents.