Gigapixel digital PCR in Giant Unilamellar Vesicles

描述(由申请人提供)：这项建议的长期目标是将DNA浓度测量的灵敏度提高1000倍。此外，我们将开发的微流控平台也将首次实现从异质样本中定向回收基因组大小的DNA片段。通过微阵列和下一代测序等技术进行的核酸分析正在改变许多生物学问题的解决方式。这些方法的力量来自于它们获得单分子细节的全系统信息的能力。然而，阻碍它们有效应用的一个主要技术障碍是，许多生物系统太复杂，无法直接测序：即使使用生物信息学算法，数十亿短序列的恐惧往往太复杂，无法拼凑成有用的信息。为了应对这一挑战，我们将开发一种技术，允许在不同种类的样本中对核酸进行定量和分类。重要的是，这种方法将利用在巨型单层细胞囊泡(GUV)中执行的特定的多重TaqMan PCR分析；这将使其比根据大小或染色属性对DNA进行分类的方法更具特异性和针对性。此外，通过分析GUV中的单个分子或细胞，我们将能够并行执行数十亿次聚合酶链式反应分析，使大量、不同种类的样本能够被筛选出来，以识别和恢复极其罕见的目标。这项基本技术将在整个生物学研究中广泛使用，并对人类健康产生直接影响，包括在疾病早期检测和测序血液中的癌症DNA，分析肿瘤细胞的遗传异质性，以及识别潜伏感染艾滋病毒的免疫细胞。具体目标1：展示耐热飞石巨单板层囊泡的微流控生成和FACS分选。我们将开发用于产生数十亿单分散飞升GUV的微流控硬件和工艺。我们还将优化FACS GUV的流程，将其分为“正”池和“负”池，并分别进入微升平板上的水井。具体目标2：优化和表征GUV-PCR和对照水滴的基准。我们将探索不同的PCR试剂来优化GUV-PCR，并将这些反应的效率与我们自己的微流体产生的油包水滴和Bio-rad QX100数字PCR机进行比较。我们将使用终点荧光、阳性反应物的比例和从反应物中回收的DNA产量来测量效率。我们还将优化用于中断GUV以访问其内容的协议。具体目标3：通过执行超过10亿次的数字GUV-PCR，并使用FAC恢复阳性分子，展示比竞争平台高1000倍的灵敏度。我们将通过执行比现有液滴技术可能进行的反应多1000倍的反应来展示GUV用于数字PCR的优越性。我们还将展示通过FACS对阳性GUV进行分类来恢复稀有DNA分子的能力。

DESCRIPTION (provided by applicant): The long term objective of this proposal is to increase the sensitivity of DNA concentration measurements by 1000X over the state of the art. In addition, the microfluidic platform we will develop to accomplish this will also enable, for the fist time, the targeted recovery of genome-size DNA fragments out of a heterogeneous sample. Nucleic acid analysis through technologies like microarrays and next-generation sequencing are transforming the way that many biological questions are addressed. The power of these approaches stems from their ability to obtain system-wide information with single molecule detail. However, a major technical barrier that often prevents their effective application is that many biological systems are too complex to be straightforwardly sequenced: Even with bioinformatic algorithms, the billions of short, sequence dreads are often too complex to piece together into useful information. To address this challenge, we will develop a technology that allows nucleic acids to be quantitated and sorted in a heterogeneous sample. Importantly, this method will utilize specific multiplexedTaqMan PCR assays performed in giant-unilamellarvesicles (GUVs); this will make it much more specific and targetable than methods that sort DNA based on size or staining properties. Moreover, by assaying individual molecules or cells in GUVs, we will be able to perform billions of PCR assays in parallel, enabling massive, heterogeneous samples to be screened to identify and recover extremely rare targets. This basic technology will be broadly useful throughout biological research and has immediate human health impacts, including for detecting and sequencing cancer DNA in the blood early in the disease, analyzing genetic heterogeneity in tumor cells, and identifying immune cells latently infected with HIV. The aims are: Specific Aim 1: Demonstrate microfluidic generation and FACS sorting of thermostable femtoliterGiant-Unilamellar Vesicles. We will develop the microfluidic hardware and processes for generating billions of monodisperse femtoliter GUVs. We will also optimize processes to FACS GUVs, both into "positive" and "negative" pools and, individually, into wells on a microliter plate. Specific Aim 2: Optimize and characterize GUV-PCR and benchmark against aqueous droplets. We will explore different PCR reagents to optimize the GUV-PCRs and benchmark the efficiency of these reactions against ones performed in aqueous-in-oil droplets generated with our own microfluidics and with the Bio-rad QX100 digital PCR machine. We will measure efficiency using endpoint fluorescence, fraction of positive reactors, and yield of DNA recovered out of the reactors. We will also optimize protocols for rupturing GUVs to access their contents. Specific Aim 3: Demonstrate 1000X greater sensitivity than competing platforms by performing over 1 billion digital GUV-PCRs, and recovery of positive molecules with FACS. We will demonstrate the superiority of GUVs for digital PCR by performing 1000X more reactions than is possible with existing droplet technologies. We will also demonstrate the ability to recover rare DNA molecules by FACS sorting the positive GUVs.