High Throughput Chromatin Immunoprecipitation on Formalin Fixed Paraffin Embedded

福尔马林固定石蜡包埋的高通量染色质免疫沉淀

• 批准号: 8750027
• 负责人: Mary Anne Jelinek
• 金额: $ 22.48万
• 依托单位: ACTIVE MOTIF, INC.
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-01 至 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8750027
• 关键词: Antibodies; Archives; Basic Science; Binding; Biocompatible Materials; Biological Assay; Biological Preservation; Chromatin; Clinical; Clinical Data; Clinical Research; Complex; Controlled Environment; CpG dinucleotide; DNA; DNA Methylation; DNA Modification Process; DNA Sequence; Development; Disease; Epigenetic Process; Feasibility Studies; Formalin; Gene Activation; Gene Expression; Generations; Genome; Genomic DNA; Genomics; Goals; Gold; Histology; Histones; Individual; Ions; Libraries; Link; Maps; Methods; Oligonucleotides; Outcome; Paraffin; Paraffin Embedding; Paste substance; Patients; Pattern; Phase; Play; Preparation; Procedures; Protocols documentation; Reaction; Reagent; Recurrence; Research; Role; Sampling; Series; Source; Staging; Technology; Testing; Tissue Sample; Tissues; Tn5 transposase; Translating; Transposase; Work; antibody conjugate; carcinogenesis; chromatin immunoprecipitation; commercialization; cost; genome-wide; histone modification; interest; new technology; next generation; next generation sequencing; novel; public health relevance; research study; sample fixation; success; therapeutic target; treatment response

DESCRIPTION (provided by applicant): Epigenetic mechanisms regulate gene expression potential and alterations in epigenetic marks, such as DNA methylation and histone modifications, have been associated with a variety of diseases. Tissue samples (both research and clinical) are often preserved by formalin fixation and paraffin embedment (FFPE), which allows for long term storage in minimally controlled environments. However there can be significant variability in the preparation of FFPE samples and the preservation conditions are harsh, making FFPE samples challenging for sophisticated downstream analyses. Clinical FFPE samples are often accompanied by valuable information including, histology, treatment course and patient outcome, thus they are an incredibly valuable source for linking basic and clinical research. The goal of this Phase I proposal is to provide the feasibility studies to use or newly developed transposase associate chromatin immunoprecipitation (TA-ChIP) to localize DNA methylation and histone modification patterns in DNA and chromatin isolated from FFPE samples. TA-ChIP uses antibody/oligonucleotide conjugates to target a transposase (Tn5) to genomic regions carrying a particular mark of interest (e.g. DNA methylation or histone modification). Upon antibody binding the transposase cuts the nearby DNA and pastes the oligonucleotides that are attached to the antibody into the DNA. Primers corresponding to these oligonucleotides can then be used for amplification and generation of next-generation libraries for genome wide sequencing. Due to the cut and paste abilities of the transposase several key steps of the traditional ChIP-seq procedure are eliminated thereby streamlining the protocol and a minimizing loss. The work described in this Phase I proposal outlines the experiments we will perform to obtain transposase compatible genomic DNA and chromatin from FFPE samples, determine optimal tagmentation conditions for genomic DNA and chromatin as well as determine whether TA-CHIP can enrich for DNA methylation and histone marks on a region specific and global scale. Aim 1 will examine DNA methylation patterns thus focusing on genomic DNA while aim 2 examines histone marks (H3K4me3, H3K27me3 and H3K9me3) and thus focuses on chromatin. Understanding the epigenetic alterations that are present in FFPE samples will allow us to not only correlate epigenetic marks with disease, but also potentially in stratifying disease state as to help predict treatment response and recurrence. If successful, these efforts will be translated into commercialization of the reagents necessary for extracting transposase compatible DNA and chromatin and the protocols necessary for these extractions as well as performing TA-ChIP ion FFPE samples. If successful, we will submit a phase II proposal in which we aim to expand the range of antibodies that could be used to study FFPE samples and also attempt to multiplex a combination of antibodies into a single ChIP reaction. Furthermore, we would also apply this technology to samples in which the preparation and preservation protocols varied widely in attempt to determine the key factors in retaining high quality DNA and chromatin during the preservation procedure.

描述（由申请人提供）：表观遗传机制调节基因表达潜力和表观遗传标记的改变，例如DNA甲基化和组蛋白修饰，与多种疾病相关。组织样本（研究和临床）通常通过福尔马林固定和石蜡包埋 (FFPE) 保存，这样可以在最低限度控制的环境中长期保存。然而，FFPE 样品的制备可能存在显着差异，且保存条件苛刻，使得 FFPE 样品对复杂的下游分析具有挑战性。临床 FFPE 样本通常附带有价值的信息，包括组织学、治疗过程和患者结果，因此它们是连接基础研究和临床研究的极其有价值的来源。该第一阶段提案的目标是提供使用或新开发的转座酶相关染色质免疫沉淀 (TA-ChIP) 定位从 FFPE 样品中分离的 DNA 和染色质中的 DNA 甲基化和组蛋白修饰模式的可行性研究。 TA-ChIP 使用抗体/寡核苷酸缀合物将转座酶 (Tn5) 靶向携带特定目标标记（例如 DNA 甲基化或组蛋白修饰）的基因组区域。抗体结合后，转座酶会切割附近的 DNA，并将附着在抗体上的寡核苷酸粘贴到 DNA 中。然后，与这些寡核苷酸相对应的引物可用于扩增和生成用于全基因组测序的下一代文库。由于转座酶的剪切和粘贴能力，传统 ChIP-seq 程序的几个关键步骤被消除，从而简化了协议并最大限度地减少了损失。该第一阶段提案中描述的工作概述了我们将进行的实验，以从 FFPE 样品中获取转座酶兼容的基因组 DNA 和染色质，确定基因组 DNA 和染色质的最佳标记条件，以及确定 TA-CHIP 是否可以在区域特定和全球范围内富集 DNA 甲基化和组蛋白标记。目标 1 将检查 DNA 甲基化模式，从而重点关注基因组 DNA，而目标 2 将检查组蛋白标记（H3K4me3、H3K27me3 和 H3K9me3），从而重点关注染色质。了解 FFPE 样本中存在的表观遗传改变不仅使我们能够将表观遗传标记与疾病相关联，而且还可能对疾病状态进行分层，以帮助预测治疗反应和复发。如果成功，这些努力将转化为提取转座酶兼容 DNA 和染色质所需的试剂以及这些提取以及执行 TA-ChIP 离子 FFPE 样品所需的方案的商业化。如果成功，我们将提交一份 II 期提案，其中我们的目标是扩大可用于研究 FFPE 样品的抗体范围，并尝试将抗体组合多重化到单个 ChIP 反应中。此外，我们还将这项技术应用于制备和保存方案差异很大的样品，试图确定保存过程中保留高质量 DNA 和染色质的关键因素。