Tools for Single Molecule and Single Cell Epigenomic Analysis

单分子和单细胞表观基因组分析工具

• 批准号: 8340779
• 负责人: HAROLD G CRAIGHEAD
• 金额: $ 57.72万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-08-21 至 2015-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8340779
• 关键词: Address; Agreement; Antibodies; Basic Science; Binding; Biological Assay; Biological Process; Cell Separation; Cells; Chemicals; Chromatin; Clinical assessments; Complex; Computer-Assisted Image Analysis; Cultured Cells; Cytosine; DNA; DNA Sequence; DNA-Binding Proteins; Data; Data Collection; Data Set; Development; Devices; Embryo; Engineering; Epigenetic Process; Fluorescent Antibody Technique; Fluorescent Probes; Generations; Genes; Genetic Materials; Genome; Goals; Health; Histones; Human Biology; Image; Immunoprecipitation; Individual; Mammals; Measures; Medicine; Methods; Methylation; Microscope; Modification; Monitor; Patients; Pharmaceutical Preparations; Population; Preparation; Process; Property; Publishing; Reporting; Research; Sampling; Sorting - Cell Movement; Source; Surface; Technology; Testing; Therapeutic; Time; base; bisulfite; chromatin immunoprecipitation; cost; disease diagnosis; drug development; epigenomics; fluorescence activated cell sorter device; fluorophore; genome-wide; histone modification; improved; induced pluripotent stem cell; information gathering; nanofluidic; next generation; operation; research study; response; single molecule; tool; trait; voltage

Epigenetic features in mammals include covalent modifications to histones, and methylation of cytosines. Their proper placement is fundamental to many biological processes. Assaying features in the epigenome is important for understanding human biology, diagnosing disease, monitoring responses to epigenome modifying drugs and facilitating development of new medicines. Such assays will also facilitate emerging therapeutics based on embryonic and induced pluripotent stem cells, which require modifying the cells to assume epigenetic states of differentiated cells. State-of-the-art assays for histone modifications use chromatin immunoprecipitation (ChIP), followed by genome wide sequencing. Methylated DNA can be identified by immunoprecipitation followed by sequencing, or by bisulfite sequencing. There are two fundamental limitations with all these approaches. First, they query only one epigenetic feature at a time. Epigenetic features arise in combinations, and those combinations rather than individual features regulate the underlying genes. Unless multiple features can be detected and measured simultaneously, it is not possible to know, with certainty, when combinations coexist on a given gene. Second, assays use populations of cells and report the average epigenetic states within the population, not the actual distribution of epigenetic states present on individual DNA molecules comprising the population. Third, ChIP often uses abundant amounts of chromatin making it impractical to assay multiple epigenetic features in rare or impossible to culture cells. In this application, we seek to develop a transforming technology, Single Chromatin molecule Analysis in Nanofluidics (SCAN) that can overcome each of these limitations and revolutionize epigenomic studies. In SCAN, chromatin molecules are bound to fluorescent probes recognizing distinct epigenetic features, then driven by voltage through nanofluidic channels where the fluorescent properties of single molecules are detected. By using multiple probes, each recognizing different features and carrying distinct fluorophores, we can directly detect their binding to individual molecules, allowing precise enumeration of multiple epigenetic features simultaneously. Our first-generation devices were operated in an analytical mode, simply counting features. Our second-generation devices were operated in a preparative mode, allowing us to sort and isolate molecules carrying defined epigenetic features. In this proposal, we seek to further develop this next generation epigenomics technology. First, we will modify the analytical device and analyte preparation to increase sample throughput by two orders of magnitude. Second, we will use the new analytical device to address selected questions in epigenomics. Third, we will use our preparative device to isolate chromatin with defined epigenetic features, sequence the DNA and compare our results to data obtained by current ChIP-seq methods.

哺乳动物的表观遗传特征包括组蛋白的共价修饰和胞嘧啶的甲基化。 它们的正确放置是许多生物过程的基础。表观基因组的分析特征是 对于理解人类生物学、诊断疾病、监测表观基因组反应非常重要 改进药物并促进新药的开发。此类检测也将促进新兴的 基于胚胎干细胞和诱导多能干细胞的疗法，需要修改细胞以 假设分化细胞的表观遗传状态。最先进的组蛋白修饰检测方法 染色质免疫沉淀 (ChIP)，然后进行全基因组测序。甲基化 DNA 可以 通过免疫沉淀随后测序或亚硫酸氢盐测序来鉴定。有两个 所有这些方法的根本局限性。首先，他们一次只查询一个表观遗传特征。 表观遗传特征以组合形式出现，这些组合而不是单个特征调节着 潜在的基因。除非可以同时检测和测量多个特征，否则不可能 确切地知道组合何时在给定基因上共存。其次，检测使用细胞群 并报告群体内的平均表观遗传状态，而不是表观遗传状态的实际分布 存在于构成群体的单个DNA分子上。第三，ChIP 经常使用大量的 染色质使得在罕见或不可能培养的细胞中测定多种表观遗传特征是不切实际的。在 在此应用中，我们寻求开发一种转化技术，单染色质分子分析 纳米流体 (SCAN) 可以克服这些限制并彻底改变表观基因组研究。在 扫描，染色质分子与识别不同表观遗传特征的荧光探针结合，然后 由电压驱动通过纳米流体通道，其中单分子的荧光特性是 检测到。通过使用多个探针，每个探针识别不同的特征并携带不同的荧光团，我们 可以直接检测它们与单个分子的结合，从而可以精确计数多个表观遗传 同时具有的功能。我们的第一代设备以分析模式运行，只需计数 特征。我们的第二代设备在准备模式下运行，使我们能够分类和隔离 携带确定的表观遗传特征的分子。在本提案中，我们寻求进一步发展这一点 一代表观基因组技术。首先，我们将修改分析设备和分析物制备 将样品通量提高两个数量级。其次，我们将使用新的分析设备 解决表观基因组学中的选定问题。第三，我们将使用我们的制备装置来分离染色质 定义表观遗传特征，对 DNA 进行测序，并将我们的结果与当前 ChIP-seq 获得的数据进行比较 方法。