The specific aim of this Phase I proposal is to establish proof of concept for development of an integrated microfluidic DNA sample prep bioMEMS device in order to greatly streamline DNA sequencing time and cost. The objectives to be addressed include: 1. Fabricating a single-use plastic microfluidic bioMEMS device (bioMEMS Card A) that removes salts and unincorporated nucleotides from a cycle sequencing reaction 2. Demonstrating that bioMEMS Card A efficiently removes these contaminants 3. Demonstrating that bioMEMS Card A can deliver a nanoliter sample volume to a glass microfluidic bioMEMS device (bioMEMS plate B) that is part of a commercial DNA sequencer 4. Comparing the sequencing results from (a) - (c) with a comparable sample prepared by ethanol precipitation and analyzed by capillary electrophoresis. This Phase I proposal is intended as part of the larger commercial effort to develop an integrated bioMEMS-based DNA sequencing system based on conventional sequencing methodologies that reduces overall reagent use by at least 100-fold. Through the microscale integration of the component steps, the system would also offer higher throughput and reduced run-to-run variability. The end result will be a commercial system that seamlessly integrates DNA sequencing from sample prep to detection on a microfluidic scale. Several groups have demonstrated microfluidic implementations of the individual steps in the overall DNA sequencing process, including amplification, filtration, and separation. To date, however, the inability to micro-integrate the various component pieces together have prevented these advances from significantly reducing the overall cost/performance of sequencing. The initial challenge is to demonstrate that multiple microfluidic-based sub-systems (designed for different chemistries, operating environments, cost points; and fabricated with different substrate materials) can be successfully integrated. For Phase I, a single use plastic bioMEMS device that replaces the purification step after the cycle sequencing reaction will be developed and demonstrated to interface with existing, multi-use glass-based microfluidic electrophoresis. The benefits of developing an automated, integrated microfluidic DNA sequencing platform include reduced reagent consumption, increased throughput, increased accuracy and faster results. Commercial applications of the product include high throughput DNA sequencing and re-sequencing for comparative and functional genomic studies as well as analysis of human genome sequence variation and diagnostics.
Thesaurus Terms: DNA, biomedical automation, biomedical equipment development, high throughput technology, nucleic acid sequence fluid flow, physical separation, plastic biotechnology, capillary electrophoresis
