Integrated microplate platform for epigenetic analysis

用于表观遗传分析的集成微孔板平台

• 批准号: 8754755
• 负责人: KAROL BOMSZTYK
• 金额: $ 23.31万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-08-15 至 2017-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8754755
• 关键词: Animal Disease Models; Antibodies; Archives; Area; Automation; Binding; Biochemistry; Biological Assay; Biological Markers; Biology; Biotechnology; Cell Count; Cell Culture Techniques; Cell Nucleus; Cells; Cellular biology; Chromatin; Clinical; Complex; DNA; DNA Modification Process; DNA analysis; DNA purification; DNA-Protein Interaction; Data Analyses; Detection; Diagnosis; Disease; Engineering; Environment; Enzymes; Epigenetic Process; Epitopes; Event; Functional disorder; Gene Expression; Genes; Genetic Transcription; Genomics; Goals; Harvest; Histones; Human; Imagery; Immunoprecipitation; Intervention; Knowledge; Mediating; Methods; Modification; Molecular Biology; Mutation; Nucleosomes; Pharmaceutical Preparations; Positioning Attribute; Precipitation; Preparation; Procedures; Process; Protein Binding; Proteins; RNA Processing; Reproducibility; Resolution; Robotics; Running; Sampling; Signal Transduction; Structure; Surface; System; Techniques; Technology; Testing; Time; Tissues; Transcription Process; Translational Research; Tube; Ultrasonography; Variant; Work; base; cell type; chromatin immunoprecipitation; chromatin modification; clinical application; computerized tools; epigenetic marker; extracellular; field study; high throughput technology; histone modification; human embryonic stem cell; improved; innovation; instrumentation; interdisciplinary approach; interest; new technology; novel; pluripotency; programs; public health relevance; receptor; response; small molecule; stem cell division; tool; transcription factor; user-friendly

DESCRIPTION (provided by applicant): Epigenetic processes interpret the genomic sequences in a cell-type and extracellular environment-dictated mode. Epigenetic information is encoded through covalent modifications of histones and DNA, nucleosome position and substitution by histone variants. Epigenetics is one of the most intensively studied fields of biology today and represents a new paradigm for the pathophysiology, diagnosis and treatment of diseases. To advance epigenetic studies, we have introduced a novel chromatin immunoprecipitation platform, Matrix ChIP, that utilizes surface-immobilized antibodies in a 96-well plate, where the entire procedure from chromatin precipitation to PCR-ready DNA purification is done on the same plate without sample transfers. This high- throughput method allows for parallel profiling of an order of magnitude greater number of chromatin and transcription events than was previously possible. Matrix ChIP is more sensitive than traditional approach to detect DNA-protein interactions of less abundant proteins such as chromatin modifiers. ChIP assay consists of three separate steps: (1) sample harvesting/chromatin fragmentation, (2) immunoprecipitation and (3) DNA purification and analysis (qPCR/sequencing). Although in the Matrix ChIP, steps (2) and (3) are greatly simplified allowing one to run hundreds of assays at a time, sample harvesting/chromatin fragmentation is done in test tubes with low efficiency and high labor intensity. This project proposes an innovative multidisciplinary approach (Dr. Matula, ultrasound/engineering, Dr. Denisenko, molecular biology, and Dr. Bomsztyk, chromatin biology/biotechnology/instrumentation) to develop a platform, PIXUL-ChIP, that will integrate highly efficient sample harvesting and chromatin shearing with immunoprecipitation in microplates for high-throughput epigenetic analysis. The following aims are proposed. Aim#1. To build a pixelated ultrasound (PIXUL) processor for high-throughput fast chromatin shearing. Aim#2. To combine sample harvesting and chromatin shearing with immunoprecipitation into an integrated microplate platform for epigenetic analysis, PIXUL-ChIP. Aim#3. To test PIXUL-ChIP as a platform for studies of epigenetic changes at selected gene loci associated with drug-mediated human embryonic stem cell (hESC) renewal and differentiation. Epigenetics is a fast growing field, but nonetheless, there are significant unmet needs that require further technological advances to facilitate exploration of epigenetic processes and exploit this knowledge in translational research and clinical applications. A highly efficient technology, PIXUL-ChIP will provide powerful means to simultaneously study multiple chromatin modifications and modifiers at gene loci in a wide range of systems, and as such will be a valuable platform for studying epigenetic processes, discovering epigenetic biomarkers, and testing novel drugs and their combinations.

描述（由申请人提供）：表观遗传过程以细胞类型和细胞外环境决定的模式解释基因组序列。表观遗传信息通过组蛋白和DNA的共价修饰、核小体位置和组蛋白变体的取代来编码。表观遗传学是当今生物学研究最深入的领域之一，代表了疾病病理生理学、诊断和治疗的新范式。为了推进表观遗传学研究，我们引入了一种新的染色质免疫沉淀平台Matrix ChIP，该平台在96孔板中利用表面固定的抗体，其中从染色质沉淀到PCR就绪的DNA纯化的整个过程在同一板上完成，而无需样品转移。这种高通量方法允许平行分析比以前可能的数量级更大的染色质和转录事件。Matrix ChIP比传统方法更灵敏地检测不太丰富的蛋白质如染色质修饰剂的DNA-蛋白质相互作用。ChIP测定由三个单独的步骤组成：（1）样品收获/染色质片段化，（2）免疫沉淀和（3）DNA纯化和分析（qPCR/测序）。虽然在Matrix ChIP中，步骤（2）和（3）被大大简化，允许一次运行数百个检测，但样品收获/染色质片段化是在试管中完成的，效率低，劳动强度高。该项目提出了一种创新的多学科方法（Matula博士，超声/工程，Denisenko博士，分子生物学和Bomsztyk博士，染色质生物学/生物技术/仪器）来开发一个平台PIXUL-ChIP，该平台将高效的样品采集和染色质剪切与微孔板中的免疫沉淀相结合，用于高通量表观遗传分析。提出了以下目标。目标1。构建一个用于高通量快速染色质剪切的像素化超声（PIXUL）处理器。目标2。将联合收割机样品收获和染色质剪切与免疫沉淀结合到用于表观遗传分析的集成微孔板平台PIXUL-ChIP中。目标3。测试PIXUL-ChIP作为研究与药物介导的人胚胎干细胞（hESC）更新和分化相关的选定基因位点表观遗传学变化的平台。表观遗传学是一个快速发展的领域，但尽管如此，仍然存在重大的未满足的需求，需要进一步的技术进步，以促进对表观遗传过程的探索，并在转化研究和临床应用中利用这些知识。PIXUL-ChIP是一种高效的技术，将为在广泛的系统中同时研究基因位点的多种染色质修饰和修饰剂提供强有力的手段，因此将成为研究表观遗传过程、发现表观遗传生物标志物和测试新药及其组合的有价值的平台。