Development of new tools for Single Cell Analysis

描述(由申请人提供)：单细胞分析正变得越来越重要，因为很明显，集合测量掩盖了细胞群体中生物学的多样性。单细胞基因分型和表型分型对于确定不同细胞类型的功能异质性是必要的，包括疾病细胞和正常细胞。该项目的总体目标是开发新的生物分析工具，用于进行高分辨率的单细胞分析。这个跨学科的项目涉及化学、分子生物学、生物化学、遗传学和生物工程，将由一个团队进行，其中包括一名高级首席研究员、一名博士后助理和两名研究生。具体目标1-开发一种高保真的单DNA分子基因分型方法，可同时分析数千个分子，并具有询问多个SNP的能力。我们计划使用光纤微孔阵列来开发一种强大、高效、快速和负担得起的方法，同时对数千个单个细胞中的单个DNA分子进行基因分型。具体目标2-开发一种分离单个细胞、捕获基因组DNA并对捕获的DNA进行基因分型的方法。在这个目标中，我们将把在特定目标1中开发的方法应用于整个细胞。数以千计的单个大肠杆菌细胞将被捕获在单独的井中，裂解，基因组DNA片断，在每个井的表面捕获DNA，然后进行基因分型。具体目标3-开发一种高分辨率的单分子分析方法，用于分析单细胞的含量。我们将开发对单个细胞内容物进行高分辨率单分子计数的方法。单个细胞将被分离、裂解，其内容物将被捕获到微孔阵列中，从而分离出单独的mRNA和蛋白质分子。然后，我们将使用各种方法来计算单个分子的数量。这些目标的成功将提供一种强大的新技术，广泛适用于生物研究的多个领域。对于生物学家来说，它提供了描述细胞群体的复杂遗传学的能力，为临床医生提供了更好的诊断机会，并将群体遗传学与治疗和临床结果的反应联系起来。对于实验者来说，它为研究肿瘤进展和对实验疗法的反应等现象提供了新的方法。对于研究衰老的研究人员，它提供了监测体细胞遗传学随年龄的变化以及影响它们的因素的能力。对于遗传学家来说，它提供了一种低成本的能力来构建遗传图谱，识别控制重组的反式作用元件，并以高分辨率监测重组产物的分子结构。我们的长期目标是将开发的技术应用于各种类型的细胞，研究基本的细胞生物学，并了解细胞之间和细胞群体的差异如何导致不同的细胞命运，并可能定义疾病。

DESCRIPTION (provided by applicant): Single cell analysis is becoming increasingly important as it is clear that ensemble measurements mask the diversity of the biology in cell populations. Single cell genotyping and phenotyping are necessary to define functional heterogeneity in varying cell types including diseased vs. normal cells. The overall goal of this project is to develop new bioanalytical tools for performing high resolution single cell analysis. This interdisciplinary project involves Chemistry, Molecular Biology, Biochemistry, Genetics, and Bioengineering and will be conducted by a team including a senior Principal Investigator, one postdoctoral associate, and two graduate students. Specific Aim 1-Develop a high fidelity single DNA molecule genotyping approach for analyzing thousands of molecules simultaneously and with the ability to interrogate multiple SNPs. We plan to employ fiber optic microwell arrays to develop a robust, efficient, fast, and affordable method of simultaneously genotyping single DNA molecules from thousands of individual cells. Specific Aim 2-Develop an approach to isolating single cells, capturing genomic DNA, and genotyping the captured DNA. In this Aim, we will apply the methods developed in Specific Aim 1 to whole cells. Thousands of single E. coli cells will be captured in individual wells, lysed, genomic DNA fragmented, the DNA captured on the surface of the individual wells, and then genotyped. Specific Aim 3-Develop a high resolution single molecule analysis method for analyzing the contents of single cells. We will develop methods for performing high resolution single molecule counting of the contents of individual cells. Single cells will be isolated, lysed, and their contents will be captured in microwell arrays such that individual mRNA and protein molecules are isolated. We will then use a variety of methods that enable us to count the individual molecules. Success of these aims will provide a powerful new technology broadly applicable to multiple areas of biological research. For biologists, it provides the ability to describe the complex genetics of cell populations, providing clinicians with improved opportunities for diagnostics, and relating population genetics with responses to therapy and clinical outcomes. For experimentalists, it provides new ways to study phenomena such as tumor progression and responses to experimental therapies. For researchers on aging, it provides the ability to monitor changes in somatic genetics with age and the factors influencing them. For geneticists, it provides a low cost ability to construct genetic maps, to identify trans-acting elements controlling recombination, and to monitor the molecular structure of recombination products with high resolution. Our long term goal is to apply the developed technology to a variety of cell types to study fundamental cell biology and to understand how cell-to-cell and cell population differences may lead to different cell fates and may define disease.