Microfluidic DNA Sequencing

描述（由申请人提供）：大多数下一代DNA测序方法集中于1)模板扩增，然后进行大规模平行合成测序，或2)单分子检测。第一种方法现在是商业上可用的，但遭受相对大量昂贵的试剂使用。后一种方法虽然尚未商业化，但在信噪比方面有缺点，因此需要更灵敏和昂贵的仪器。为了避免这些缺点，我们将使用基于微滴的微流体来测序DNA。通过使用微流体，我们将测序DNA所需的试剂量限制在几毫升以下，同时仍然保留扩增模板的能力，从而使我们能够使用相对便宜和强大的检测。短探针的杂交将在微流控液滴中通过荧光偏振的变化来检测，这种变化区分了结合寡核苷酸和自由寡核苷酸。这消除了对分离相检测杂交的要求。该方法简单，不需要酶。在第一阶段，我们将描述一个简单的平台和重测序方法，将在未来的第二阶段项目中进行扩展，使人类基因组测序的成本低于1000美元。

DESCRIPTION (provided by applicant): 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.