|Genome Sequencing Technologies and Resources
DOE Human Genome Program Contractor-Grantee Workshop
52. Development of Flowthrough Genosensor Chips
Mitchel J. Doktycz and Kenneth L.
A flowthrough genosensor chip is under development at ORNL. The core of this technology is a microchannel hybridization array, containing numerous specific DNA sequences, immobilized within individual cells of densely packed straight, smooth channels traversing a thin silicon or glass substrate. When a nucleic acid sample is labeled and passed through the microchannel genosensor chip, hybridization occurs at porous cells bearing immobilized DNA probes complementary to the target sequence. The quantitative binding pattern reflects the relative abundance of specific target sequences within the nucleic acid analyte. The flowthrough chip configuration has several important advantages over flat surface DNA chips being developed elsewhere: faster hybridization kinetics, superior binding capacity, improved ability to analyze dilute solutions of nucleic acids, including both strands of a heat-denatured PCR fragment.
Related technology for taking advantage of the benefits of the flowthrough genosensor include the development of micromachining techniques for the construction of flowthrough silicon chips to complement those constructed using channel glass. A customized robotic spotting system has been developed that includes a high resolution positioning system, sapphire dispensing tips for touch-off dispensing, and, more recently, solenoid-controlled ink jets for remote droplet delivery. A prototype fluidics system has been developed that involves syringe pump-driven fluid flow, a custom chip holder attached to the stage of a Zeiss Axiovert fluorescence microscope and a CCD camera for real-time quantitative detection of hybridized fluorescent-labeled strands. A software package for intelligent selection of oligonucleotide probes for a given chip application has been developed.
The flowthrough genosensor system is now being used to develop applications in the areas of genotyping and mRNA profiling, in collaboration with various laboratories. Gene expression profiling in mammalian systems, including mouse and sheep, is being pursued as well as bacterial systems for evaluating expression patterns in soil microorganisms as an indicator of genotoxic response in the environment. Another application being developed is high throughput genotyping. In this work miniature flowthrough genosensors are used to simultaneously analyze numerous single nucleotide and short insertion-deletion polymorphisms. In another application area, the ultrahigh surface area of channel glass is being exploited to create arrays of "microreactor cells" containing immobilized BAC DNAs, for use in repetitive reactions needed for genome mapping and sequencing, including cycle sequencing reactions, PCR, and hybridization mapping of expressed sequences to their genomic clones.
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