Epigenomic analysis on a nanoscale device

描述（由申请人提供）：在本申请中，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。该方法建立在我们过去开发的一项技术的基础上，该技术使我们能够通过使用飞图数量的DNA以每分钟超过4,000分子的速度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嵌入染料标记的噬菌体DNA标记，用于高通量单DNA分子分析的原理验证测试。在三个目标中，我们建议以逐步和系统的方式扩展我们现有的技术，以：(a)同时分析哺乳动物染色质中DNA和多个表观遗传标记的甲基化；(b)使用从极低丰度来源分离的染色质，如着床前胚胎和激光显微解剖肿瘤；(c)对携带特定标记组合的DNA进行排序和恢复，以便随后进行高通量DNA测序。如果成功，我们的努力将彻底改变表观基因组研究，使单个细胞的整个表观基因组分析成为可能；将表观遗传修饰检测的灵敏度提高了几个数量级；促进同时监测多种表观遗传修饰；并提供表观遗传状态与基因表达能力之间的功能关联。这项技术将大大提高参考表观基因组作图中心的能力。

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.