Third Wave's Invader: 99.7% Accuracy in Large, Multicenter Pharmacogenomic Study
SNPs are the most common genetic variations among individuals, occurring at an estimated frequency of at least 1% in the population and affecting approximately 1 in 1000 nucleotides or as many as 1 million bases per genome. The ability to compare genomic variation between individuals has accelerated the discovery of genes associated with common diseases. In addition, SNP analysis is the tool that could allow healthcare to be personalized for each individual patient.
The Invader assay detects specific mutations and SNPs in unamplified genomic DNA. The accuracy comes from the extreme structure specificity of the enzymes, combined with the sequence specificity of the probe hybridization. Mismatches at the position of overlap between the probes will block cleavage, allowing this method to be used to discriminate between variants that differ by a single base.
"PCR-RFLP, the current gold-standard for genotyping, lacks the accuracy and ease of use to allow large-scale genotyping studies," commented Brian Dawson, director of the Molecular Diagnostics Laboratory at the University of Texas Southwestern Medical center (Dallas). "As the number of genetic markers and the number of samples to be analyzed per marker rapidly increases, there is a significant need to adopt a highly accurate and easier-to-use gold standard such as the Invader technology."
"The benefit of Third Wave's Invader technology is its ability to very accurately detect genetic variations directly from unamplified genomic DNA using a simple, homogeneous format that does not require PCR amplification," said Martin Hessner, director of product development, DNA Diagnostic Laboratories at The Blood Center of Southeastern Wisconsin (Milwaukee). "Third Wave's Invader technology can be easily integrated into routine clinical laboratory practice, and the format allows automation not possible with PCR methods such as allele-specific PCR and PCR-RFLP."
"The Invader technology prompted us to readdress our need to outsource DNA-based testing, which was until recently being performed elsewhere using PCR-based methods," said Marlies Ledford, technical director, Coagulation Laboratory, University of Miami
School of Medicine. "The accuracy, ease of use and cost efficiencies of the Invader technology has given us the opportunity to bring DNA-based testing in-house, using Third Wave's analyte-specific reagents. I believe this has increased the quality and timeliness of care that we provide to our patients."
More than one-third of the 2,530 samples in the multi-center study were genotyped for the Factor V mutation (G1691A), and slightly less than one-third of these samples were genotyped for each of the Factor II (G20210A) and MTHFR (C677T) mutations. The combined results for the three mutations from the six participating laboratories were 2,117/2,122 homozygous normal, 345/348 heterozygous and 60/60 homozygous mutant, or 99.7% (2,522/2,530) concordant overall between the results of the Invader technology and the multiple PCR-based methods utilized.
"The need for accuracy approaching 100% is obvious in both the research and clinical setting when one is looking at multiple genetic variations," said Lance Fors, president and CEO of Third Wave. "However, what is not fully appreciated is that there may be an absolute requirement for routine accuracy of at least 99.5% to permit decoding and direction of treatments for common diseases when multiple genetic variations need to be analyzed."
A high degree of accuracy will be essential for studies to detect genetic variations associated with common diseases, which will involve the analysis of thousands of genetic variations per individual. For example, in a test panel involving six genetic variations, the overall panel accuracy for a technology having a 90% accuracy per result would be 53%, [(0.90)6 = 0.53] as compared to 97% [(0.995)6 = 0.97] for a technology having 99.5% accuracy per result.
How It Works
Invader detects specific DNA and RNA sequences by using structure-specific "cleavase" enzymes to cleave a complex formed by the hybridization of overlapping oligonucleotide probes. Elevated temperature and an excess of one of the probes enable multiple probes to be cleaved for each target sequence present without temperature cycling. Cleaved probes then direct cleavage of a labeled probe.
Third Wave's Invader assay kit
The secondary probe oligonucleotide can be 5'-end labeled with fluorescein that is quenched by an internal dye. Upon cleavage, the fluorescein labeled product may be detected using a standard fluorescence plate reader. These sequential reactions produce 106 to 107 labeled cleavage products per target sequence per hour.
About Third Wave
Third Wave has existing agreements with major pharmaceutical companies to use its Invader technology for large-scale pharmacogenomics studies and emerging therapeutic selection applications in the clinic. Third Wave also has collaborations with leading research institutes, including the Sanger Centre and Stanford, Cambridge, and Oxford Universities, which are using Invader technology in a wide range of genome research and clinical applications. Third Wave also markets analyte specific reagents and assay controls, based on the Invader technology, for use by clinical laboratories as part of internally validated "home brew" assays.
For more information: Lance Fors, CEO, Third Wave Technologies, 502 South Rosa Rd., Madison, WI 53719. Tel: 608-273-8933. Fax: 608-273-8618.
Angelo DePalma