METHYLMAP: A TECHNOLOGY TO ANALYZE PROMOTER METHYLATION IN MICRODISSECTED CELLS

甲基图：一种分析微解剖细胞中启动子甲基化的技术

• 批准号: 7571328
• 负责人: Robi D Mitra
• 金额: $ 38万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-20 至 2012-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7571328
• 关键词: Biological Sciences; Cataloging; Catalogs; Cell Count; Cells; Complex; DNA; DNA Methylation; DNA Sequence; Depth; Development; Epigenetic Process; Gene Expression Regulation; Genes; Heterogeneity; Homeostasis; Lasers; Lead; Location; MLH1 gene; Malignant Neoplasms; Measurement; Measures; Methylation; Mutation; Onset of illness; Pattern; Play; Public Health; Research; Role; Sampling; Scientist; Somatic Cell; Technology; Time; Tissues; Tumor Biology; bisulfite; cell type; high throughput technology; insight; interest; next generation; promoter; single molecule; tool; tumor; tumorigenesis

DESCRIPTION (provided by applicant): Epigenetic marks such as DNA methylation play important roles in mammalian development and the onset of disease. Our ability to study methylation in complex tissues is limited because existing tools measure the average methylation of a large number of cells. Since most tissues contain many different cell types, these bulk measurements are inaccurate. We hope to create an entirely new way to analyze methylation in complex tissues. We propose MethylMap, a high-throughput technology that combines multiplex amplification, sample-specific DNA barcodes, and next- generation sequencing to analyze methylation in laser capture micro-dissected cells. Our research plan consists of two specific aims. First, we will demonstrate the multiplexed amplification and single-molecule sequencing of 5000 promoters from a single sample of bisulfite treated DNA. We will use this technology to catalogue epigenetic mutations that arise during tumorigenesis. Next, we will use the 454 FLX sequencing platform to bisulfite sequence 200 promoters from laser capture microdissected cells arrayed in 96 well plates (or 19,200 promoters per plate). Each sample will be barcoded with a DNA tag to connect the measured methylation pattern with the location of the cells. We will build on preliminary results that demonstrate 1) multiplexed amplification of 90 loci from a single sample, 2) deep single molecule bisulfite sequencing of the MLH1 promoter in tumors using the 454 Life Sciences FLX sequencer, and 3) the use of sample-specific DNA barcodes with next-generation sequencing technology. MethylMap will have many applications: it will be used to molecularly classify cells in complex tissues, to determine the lineages of somatic cells, and to probe the role of aberrant methylation in tumorigenesis. We believe MethylMap will become an important tool for scientists interested in development, tissue homeostasis, and tumor biology. PUBLIC HEALTH RELEVANCE: DNA methylation is an important mechanism used by the cell to control gene regulation. Recent studies have shown that DNA methylation is critical for mammalian development, and if DNA methylation is misregulated, it can lead to cancer. Current tools to study DNA methylation are inadequate because they cannot effectively analyze complex tissues that contain many different types of cells. We propose to develop MethylMap, a technology that will make it possible to analyze the methylation patterns of cells in complex tissues for the first time. This tool will provide new insights into how tissues form, and how cancers arise.

描述（由申请人提供）：表观遗传标记，如DNA甲基化在哺乳动物发育和疾病发病中起重要作用。我们研究复杂组织中甲基化的能力是有限的，因为现有的工具只能测量大量细胞的平均甲基化。由于大多数组织包含许多不同的细胞类型，这些体积测量是不准确的。我们希望创造一种全新的方法来分析复杂组织中的甲基化。我们提出MethylMap，这是一种高通量技术，结合了多重扩增，样品特异性DNA条形码和下一代测序来分析激光捕获微解剖细胞中的甲基化。我们的研究计划包括两个具体目标。首先，我们将演示从亚硫酸氢盐处理过的DNA样品中提取5000个启动子的多重扩增和单分子测序。我们将使用这项技术对肿瘤发生过程中出现的表观遗传突变进行分类。接下来，我们将使用454 FLX测序平台对排列在96个孔板（或每个孔板19,200个启动子）的激光捕获微解剖细胞中的200个启动子进行亚硫酸测序。每个样本将用DNA标签进行条形码，以将测量的甲基化模式与细胞的位置联系起来。我们将以初步结果为基础，证明1)从单个样品中多重扩增90个基因座，2)使用454 Life Sciences FLX测序仪对肿瘤中MLH1启动子进行深度单分子亚硫酸盐测序，以及3)使用样本特异性DNA条形码与下一代测序技术。MethylMap将有许多应用：它将用于复杂组织中细胞的分子分类，确定体细胞的谱系，并探索异常甲基化在肿瘤发生中的作用。我们相信MethylMap将成为对发育、组织稳态和肿瘤生物学感兴趣的科学家的重要工具。