Gigapixel digital PCR in Giant Unilamellar Vesicles

描述（由申请人提供）：该提案的长期目标是将DNA浓度测量的灵敏度提高1000倍于现有技术水平。此外，我们将开发的微流控平台也将首次实现从异质样品中靶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。这些方法的强大之处在于它们能够获得具有单分子细节的全系统信息。然而，通常阻碍其有效应用的一个主要技术障碍是，许多生物系统过于复杂，无法直接测序：即使使用生物信息学算法，数十亿个简短的序列数据也往往过于复杂，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，允许在异质样品中对核酸进行定量和分选。重要的是，该方法将利用在巨单层囊泡（GUV）中进行的特异性多重TaqMan PCR检测;这将使其比基于大小或染色特性对DNA进行分选的方法更具特异性和靶向性。此外，通过分析GUV中的单个分子或细胞，我们将能够并行进行数十亿次PCR检测，从而能够筛选大量异质样品，以识别和回收极其罕见的靶标。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，分析肿瘤细胞中的遗传异质性，以及识别潜伏感染HIV的免疫细胞。具体目标1：演示热稳定性飞秒巨单层囊泡的微流体生成和FACS分选。我们将开发微流体硬件和工艺，以产生数十亿个单分散毫微微升GUV。我们还将优化FACS GUV的过程，将其分为"阳性"池和"阴性"池，并分别进入微升板上的威尔斯孔。 � 具体目标2：优化和表征GUV-PCR，并针对水性液滴进行基准测试。 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和Bio-rad QX100数字PCR仪产生的油包水液滴进行比较。我们将使用终点荧光、阳性反应器的分数和从反应器中回收的DNA的产率来测量效率。我们还将优化破裂GUV以获取其内容物的协议。 � 具体目标3：通过执行超过10亿次数字GUV-PCR和使用FACS回收阳性分子，证明灵敏度比竞争平台高1000倍。我们将通过执行比现有液滴技术多1000倍的反应来证明GUV用于数字PCR的优越性。我们还将证明通过FACS分选阳性GUV回收稀有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.