Direct molecular analysis without amplification
The Trilogy™ Platform (US Genomics) is founded on the belief that direct analysis of single biological molecules is the key to the next generation of revolutionary technologies. US Genomics is developing technologies that can directly analyze individual molecules of DNA, RNA and proteins, without PCR amplification. The process of direct analysis by the Trilogy platform begins with the isolation of target material (DNA, RNA or protein) from a biological source, followed by fluorescently tagging the sample material at specific sites of interest (e.g. a nucleotide sequence motif or protein epitope). The sample is then injected into the microfluidic system of the Trilogy Instrument, and the sample passes through an interrogation region consisting of several laser spots. Each molecule is detected by the laser excitation of the fluorescent tags on the molecule. Thousands of molecules pass through the system per minute.
Trilogy Analysis replaces traditional molecular biology techniques, hampered by the need for amplification and bulk fluorescence, with the accuracy and sensitivity of direct measurements based on single molecules. Direct analysis on the Trilogy platform utilizes color coincidence counting to detect and quantify individual biomolecules that have been fluorescently labeled, then directed by proprietary microfluidics to the Trilogy’s multi-color laser interrogation and detection region. Individual molecules are monitored by color fluorescence, and color coincidence is used to distinguish and quantify true molecular events, lending the system high specificity and strong signal-to-noise. Additional advantages of the platform include:
1. Sensitivity in the femtomolar range.
2. No need for amplification (e.g. PCR) or enzymatic procedures, eliminating a major source of cost and bias.
3. Small sample material requirements.
4. Flexibility across sample types (e.g. DNA, RNA and protein) and assays.
Trilogy Analysis is compatible with a wide range of sample types and assays. Quantification of molecules of interest such as RNA transcripts is accomplished by direct dual-labeling of RNA with fluorescently labeled oligonucleotides, eliminating the need for reverse transcription or PCR. Detection of specific nucleotide sequences such as SNP polymorphisms is achieved through probe hybridization or an extension reaction. And interactions between two fluorescently labeled molecules (e.g. protein-protein, protein-DNA, protein-small molecule) can easily be detected and quantified at the molecular level, making biochemical characterization of such interactions a rapid, accurate and convenient process.
In DirectLinear™ Analysis, DNA molecules up to megabases in length are tagged with specific fluorophores and pass through a proprietary microfluidics system which stretches them to their full length, causing them to pass through and be read by the laser excitation and detection region in a linear fashion. The data generated is equivalent to a genetic 'barcode' representing the spatial map of the fluorescent tags along the DNA. Each genomic barcode is unique to a specific individual or organism.
DirectLinear™ Analysis has numerous potential applications in life science research and drug discovery and development. Entire genomes of novel organisms can be mapped nearly instantaneously, inviting comparison with known genomes and allowing researchers to focus on conserved regions or novel genomic features. Genetic differences between two samples or populations can readily be detected by comparing differences in barcode patterns, allowing the rapid identification of polymorphisms associated with disease or adverse drug response. Rapid genomic mapping of microbial organisms will have great utility in research and diagnostics in infectious disease, as well as in biodefense. Finally, rapid, low-cost access to each person’s genomic information is the key to enabling molecular diagnostics and, ultimately, personalized medicine.
Related Links The PCR Jump Station | Information and links on PCR | The PCR Gateway | The PCR Directory