Microfluidic DNA Sequencing

描述(申请人提供)：大多数下一代DNA测序方法侧重于1)模板扩增，然后大规模平行合成测序，或2)单分子检测。第一种方法现在可以在商业上使用，但存在相对大量昂贵的试剂使用问题。后一种方法虽然还没有商业化，但在信噪比方面存在劣势，因此需要更灵敏和昂贵的仪器。为了避免这些缺点，我们将使用基于液滴的微流控来对DNA进行测序。通过使用微流体，我们将DNA测序所需的试剂量限制在几毫升以下，同时仍然保留了放大模板的能力，从而使我们能够使用相对便宜和强大的检测。通过区分结合寡聚和游离寡聚的荧光偏振的变化，将在微流控液滴中检测到短探针的杂交。这消除了检测杂交需要分离相的要求。该方法简单，不需要酶。在第一阶段，我们将描述一种简单的平台和重新测序方法，该方法将在未来的第二阶段项目中进行扩展，以实现以低于1000美元的价格进行人类基因组测序。 与公共卫生相关：拟议的第一阶段基于液滴的微流体仪器将每秒产生、选择性地合并和分析10,000多个水滴。我们希望利用这一能力在微流体平台上实施DNA测序方法。我们把这个项目分成了两个阶段。第一阶段是原则证明。在第一阶段中，我们演示了生化分析在液滴中工作，也证明了我们的检测器工作。在第二阶段，我们将扩大这一过程，以制造一台能够以不到1000美元的价格对人类基因组进行测序的机器。

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.