SBIR-STTR Award

Current high-throughput DNA sequencing systems can produce sequence data at a theoretical rate of approximately 29 megabases per year. Capillary Array Electrophoresis (CAB) systems can increase the DNA sequencing throughput by an order-of-magnitude over currently available systems. A flowable separation medium is one of the key components of the CAB system, and improvements in separation media significantly lower the cost of a CAB system. The moderate-to-high viscosities of the separation media used by previous researchers makes it a formidable task to design a pressurized capillary refilling station for a commercial CAB system. The goal of Phase I of this proposal is to develop low-viscosity media which will enable single base resolution out to 1000 bases. Using low-viscosity separation media will simplify the pressure-vessel design issues of the CAB system, resulting in a substantial reduction in cost. Use of such high-throughput CAB systems will revolutionize large-scale sequencing projects, and substantially reduce the cost per base. Phase II studies will extend the utility of low-viscosity and high-resolution media with less expensive dynamically-coated capillaries by eliminating the need for high-cost covalently-coated capillaries. This will further reduce the cost per system and per base sequences. PROPOSED COMMERCIAL APPLICATIONS: A CAE system has significant potential to improve the U.S. economic growth and productivity by accelerating the rate that new genetic information is generated. Reliable, cost-efficient sequencing and genetic typing will have significant impact on Human Genome Project, healthcare, agribusiness, forensic analysis, and pharmaceutical industry.

Thesaurus Terms:
biotechnology, capillary electrophoresis, fluid flow, nucleic acid sequence, polymer confocal scanning microscopy, fluidity, fluorescence, siliconNational Institute of Occupational Safety and Health (NIOSH)

The goal of the Phase II grant is to develop a DNA sequencing matrix for capillary electrophoresis that achieves very long read lengths (1000 bases) in a semi-automatic, high-throughput environment. We will use the analysis tools developed in Phase I to conduct an advanced investigation of the behavior of linear polymers as sieving matrices for DNA sequencing. We will concentrate on identification and elimination of the factors that contribute to peak broadening. We will investigate the effect of the molecular mass distribution of the sieving polymers on selectivity in both Ogston and reputation modes, as well as the effect of chemical composition of the polymers on both selectivity and separation efficiency. Further, we will optimize the composition of the background electrolyte to improve both selectivity and separation efficiency. This effort will lead to a sieving matrix formulation that supports long sequencing readlengths in a semi-automatic, high-throughput sequencing system. PROPOSED COMMERCIAL APPLICATIONS: Current commercial capillary sequencers produce high-accuracy sequencing read lengths averaging about 450 bases and rarely exceeding 600 bases. This work has the potential to double the average read length. This would reduce the cost of EST sequencing by half, and would be more significant for genome sequencing, since long read lengths greatly facilitate sequence assembly and closure. The resulting matrix could be quickly supplied for commercial use on the MegaBACE 1000 DNA Sequencing System.

Thesaurus Terms:
biomaterial development /preparation biomaterial evaluation