Technologies for visualizing the genome in situ

项目概要/摘要 目标：关于染色体组织在遗传和基因表达中的作用的问题- 锡永提出了许多技术挑战。染色体DNA不仅是细胞中最大的生物分子， 它由以细胞与细胞不同的方式独立折叠的片段组成。此外，前 二倍体生物的染色体有X和Y，它们是成对的同源染色体，很难区分。 通过成像进行着色。本申请提出了一套工具，用于跟踪整个染色体的路径- 一些在单细胞水平上的序列和同源物特异性的方式，使用宽场，共聚焦， 以及超分辨率显微镜。作为工具的一个方面是寡核苷酸文库（oligos）， 虽然可再生，但价格昂贵，这样的图书馆将被设计为广泛有用， 可以在实验室之间自由共享。简而言之，这一建议旨在提供新的工具， 万斯对染色体功能和染色体组织结构之间关系的理解， 是的。 健康相关性：细胞核内基因组的组织和基因组的进展 这种通过发展形成的组织对基因表达有着深远的影响，因此， 发育和疾病;染色体拓扑结构和染色体间相互作用影响增强子- 启动子相互作用，整体沉默，染色体稳定性和修复，染色体分离， 甚至是继承权因此，基于单细胞成像的研究阐明了基因组如何 将大大有助于我们进一步了解和治疗疾病。 创新：拟议的目标主要集中在新技术的开发上 因此，它们有希望使研究和发现，否则可能是不可能的。 特别是，他们的目标是通过提高探针的有效性来促进染色体原位成像 和探头设计，提高数据采集速度和收集的数据量， 图像分辨率通过这种方式，他们可以为社区提供工具， 基因组，如人类的基因组，更容易进行分析。简而言之，这些目标致力于： 目标1优化寡聚体 目的2使用Oligopaints、OligoSTORM和OligoDNA-PAINT追踪染色体 目的3利用OligoFISSEQ追踪染色体

PROJECT SUMMARY/ABSTRACT Goals: Questions regarding the role of chromosome organization on inheritance and gene expres- sion raise many technical challenges. Not only is chromosomal DNA the largest biomolecule in the cell, it is comprised of segments that fold independently in ways that vary from cell to cell. Furthermore, ex- cepting the X and Y, chromosomes of diploid organisms come in pairs of homologs that defy easy dis- tinction via imaging. This application proposes a suite of tools for tracing the path of entire chromo- somes at the single cell level in a sequence- and homolog-specific fashion using widefield, confocal, as well as super-resolution microscopy. As one aspect of the tools are libraries of oligonucleotides (oligos) which, while renewable, are expensive, such libraries will be designed to be broadly useful so that they can be shared freely among laboratories. In brief, this proposal aims to provide new tools that will ad- vance our understanding of the relationship between chromosome function and chromosome organiza- tion. Health relatedness: The organization of the genome within the nucleus and the progression of that organization through development has profound consequences for gene expression and, thus, development and disease; chromosome topology and interchromosomal interactions affect enhancer- promoter interactions, global silencing, chromosome stability and repair, chromosome segregation, and, thus, even inheritance. As such, single-cell imaging-based studies that elucidate how the genome is organized will do much to further our understanding and treatment of disease. Innovation: The proposed aims are focused heavily on the development of new technologies and, thus, they hold promise for enabling studies and discoveries that might not otherwise be possible. In particular, they aim to facilitate the imaging of chromosomes in situ by improving probe effectiveness and probe design, increasing speed of data acquisition and quantity of data collected, and enhancing image resolution. In this way, they may provide the community with tools that can render complex genomes, such as those of humans, more accessible for analysis. In brief, the aims are dedicated to: Aim 1 Optimizing Oligopaints Aim 2 Tracing chromosomes using Oligopaints, OligoSTORM, and OligoDNA-PAINT Aim 3 Tracing chromosomes using OligoFISSEQ