Most next-generation DNA sequencing methods have focused on either 1) template amplification followed by massively-parallel sequencing-by-synthesis, or 2) single molecule detection. The first method is now commercially available but suffers from relatively large volumes of expensive reagent usage. The latter method, although not yet commercially available, will have a disadvantage in signal-to-noise and therefore require more sensitive and expensive instrumentation. To avoid these disadvantages, we will use droplet-based microfluidics to sequence DNA. By using microfluidics we limit the amount of reagent required to sequence DNA to less than several milliliters, while still retaining the ability to amplify the template that thereby enables us to use relatively inexpensive and robust detection. Hybridization of short probes will be detected in microfluidic droplets by a shift in fluorescence polarization that distinguishes between bound and free oligo. This removes the requirement for a separation phase to detect hybridization. The method is simple and does not require enzymes. In Phase I we will describe a simple platform and resequencing method that will be scaled in a future Phase II project to enable human genome sequencing for under $1000. Public Health Relevance: The proposed Phase I droplet-based microfluidics instrument will generate, selectively merge and analyze over 10,000 aqueous droplets per second. We want to leverage this capability to implement a DNA sequencing method on a microfluidics platform. We have broken the project up into two phases. Phase I is proof of principle. In Phase I we demonstrate that the biochemical assay works in drops and also that our detector works. In Phase II we will scale the process to build a machine that will be able to sequence a human genome for less than $1000.
The proposed Phase I droplet-based microfluidics instrument will generate, selectively merge and analyze over 10,000 aqueous droplets per second. We want to leverage this capability to implement a DNA sequencing method on a microfluidics platform. We have broken the project up into two phases. Phase I is proof of principle. In Phase I we demonstrate that the biochemical assay works in drops and also that our detector work. In Phase II we will scale the process to build a machine that will be able to sequence a human genome for less than $1000.
