Even small numbers of single-nucleotide polymorphisms (SNPs) can work as predictive tools to assist clinical decisions, and this is the area that interests Third Wave Technologies. Based in Madison, Wisconsin, the company has set low-volume SNP-genotyping tests for individual diagnostics as its priority.
“We started out in the cardiovascular arena and helped to create the field for 'economy-class syndrome' mutation detection,” explains chief executive Lance Fors. Third Wave developed a reasonably priced test for mutations in the blood-clotting factors V and II that can trigger venous blood clots in the brain and legs. Women who carry the factor V mutation (V Leiden) and are also taking contraceptive pills are at 100 times greater risk of deep-vein thrombosis and stroke, and today an increasing number of women taking contraceptives are tested for these variants. “It's moving towards mainstream medicine,” says Fors. “An individual test for a specific SNP may turn into a $10- or $30-million market.”
Low false negatives and low false positives are the winning solution in diagnostics, Fors adds. Third Wave's platform uses the firm's Invader RNA Assay. Two probes are designed to overlap or 'invade' each other to make a particular three-dimensional structure. If the mutation is present, there is an invasion and the structure is formed. A nuclease then recognizes this overlapping complex and cuts it. With 'normal' DNA, the probes cannot invade and the DNA remains intact, Fors explains. Structure-specific cleavage is far more precise than conventional methods based on the polymerase chain reaction (PCR), so mistakes are very rare, he points out.
The Invader technology is a one-step assay that simply requires the sample to be added. “Some of our clinical customers could never adopt PCR, because they couldn't afford the equipment or the infrastructure required to minimize contamination,” Fors says.
Invader technology can scale up to detecting hundreds of thousands of SNPs, as its fluorescence readout is readily adaptable for high-throughput. It was used to screen 768 Japanese cardiovascular patients for the presence of 92,000 SNPs located in genes that are thought to confer a risk of cardiovascular disease, and to identify a candidate gene associated with susceptibility to heart attack on chromosome 6p21 (K. Ozaki et al. Nature Genet. 32, 650–654; 2002).
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Risk assessment. Nature 422, 919 (2003). https://doi.org/10.1038/422919a