Epigenomic analysis on a nanoscale device

描述(由申请人提供)：在这项申请中，我们寻求开发一种转化技术，将彻底改变表观基因组学研究。这种方法建立在我们过去开发的一项技术的基础上，该技术允许我们通过使用飞克级DNA以每分钟超过4000个分子的速度进行荧光成像，来表征复杂混合物中的单个DNA分子。这些先前的荧光成像研究使用插入染料标记的噬菌体DNA标记进行高通量单DNA分子分析的原理验证测试。在三个目标中，我们建议逐步和系统地扩展我们的现有技术，以(A)在哺乳动物染色质中同时分析DNA甲基化和多个表观遗传标记；(B)使用从极低丰度来源(如植入前胚胎和激光显微解剖的肿瘤)分离的染色质来实现这一点；以及(C)对携带特定标记组合的DNA进行分类和回收，以便随后进行高通量DNA测序。如果成功，我们的努力将使表观基因组研究发生革命性的变化，使单细胞的整个表观基因组图谱成为可能；将表观遗传修饰检测的灵敏度提高几个数量级；促进对多个表观遗传修饰的同时监测；并提供表观遗传状态和基因表达能力之间的功能关联。这项技术将极大地增强参考表观基因组测绘中心的能力。 与公共卫生相关：在过去的20年里，非常清楚的是，基因组的表观遗传改变可以像基因组的突变一样深刻地影响发育和健康。最具戏剧性的例子之一是，p16抑癌基因启动子上的DNA甲基化在沉默基因方面与基因本身的突变一样有效，这两种事件都有助于结直肠癌的发生和发展[1]。重要的是，与突变不同，p16的表观遗传沉默可以在药物上逆转，具有潜在的治疗益处[2]。这项提案寻求开发一种革命性的新工具，用于在极少量的材料中同时和全基因组范围内评估多种表观遗传修饰。如果成功，这将极大地增加目前正在形成的参考表观基因组图谱中心可能进行的表观遗传学分析。这项技术将为这些中心的发现任务及其对公共健康的影响带来巨大好处。

DESCRIPTION (provided by applicant): In this application, we seek to develop a transforming technology that will revolutionize epigenomic studies. The approach builds upon a technology we have developed in the past that allowed us to characterize individual DNA molecules in a complex mixture by fluorescence imaging at a rate exceeding 4,000 molecules per minute using femtogram quantities of DNA. These previous fluorescence-imaging studies used phage DNA markers labeled with intercalating dyes for a proof-of-principle test for high throughput single DNA molecule analysis. In three Aims, we propose to extend our existing technology in a stepwise and systematic way, to (a) analyze methylation on DNA and multiple epigenetic marks simultaneously in mammalian chromatin; (b) do this using chromatin isolated from extremely low abundance sources, such as preimplantation embryos and laser microdissected tumors; and (c) sort and recover DNAs carrying specific combinations of marks for subsequent high throughput DNA sequencing. If successful, our efforts will revolutionize epigenomic studies by enabling whole epigenome profiling of single cells; increasing the sensitivity of epigenetic modification detection by several orders of magnitude; facilitating simultaneous monitoring of multiple epigenetic modifications; and providing functional correlates between epigenetic states and gene expression competence. This technology will greatly enhance the capabilities of the Reference Epigenome Mapping Centers. PUBLIC HEALTH RELEVANCE: It has become abundantly clear during the past 20 years that epigenetic alterations to the genome can influence development and health as profoundly as mutagenesis of the genome. One of the most dramatic examples is the fact that methylation of DNA at the promoter of the p16 tumor suppressor is as effective at silencing the gene as mutations to the body of the gene itself and that both events contribute to the development and progression of colorectal cancer [1]. Importantly, unlike mutations, epigenetic silencing of p16 can be reversed pharmacologically, with potential therapeutic benefit [2]. This proposal seeks to develop a revolutionary new tool for assessing multiple epigenetic modifications, simultaneously and genome-wide, in vanishingly small quantities of material. If successful, this will vastly increase the epigenetic analyses possible in the now-forming Reference Epigenome Mapping Centers. This technology will be of enormous benefit to the discovery mission of these Centers and their impact on public health.