Technologies for visualizing the genome in situ

项目摘要/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 Sion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， 它由独立折叠的片段组成，这些片段的折叠方式因细胞而异。此外，前- 除了X和Y，二倍体生物的染色体是成对的同源染色体，不容易分离 通过成像进行消退。这个应用程序提出了一套工具来追踪整个染色的路径- 在单个细胞水平上以序列和同源基因特有的方式使用广域、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(寡核苷酸)的文库 这些库虽然可再生，但价格昂贵，这样的库将被设计为广泛使用，以便它们 可以在实验室之间自由共享。简而言之，这项提议旨在提供新的工具，使- 我们对染色体功能和染色体组织之间的关系的理解-- 提顿。 与健康相关：基因组在细胞核内的组织和进展 这种组织通过发展对基因表达产生了深远的影响，因此， 发育和疾病；染色体拓扑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，染色体稳定和修复，染色体分离， 因此，甚至还有继承权。因此，基于单细胞成像的研究阐明了基因组是如何 组织起来将极大地促进我们对疾病的理解和治疗。 创新：拟议的目标主要集中在新技术的开发上 因此，它们有望推动研究和发现，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效率来促进染色体的原位成像 和探头设计，提高了数据采集的速度和数据量，增强了 图像分辨率。通过这种方式，他们可以为社区提供可以呈现复杂的工具 基因组，例如人类的基因组，更容易进行分析。简而言之，这些目标致力于： 目标1优化齐聚色漆 目的2用寡核苷酸、寡核苷酸和寡核苷酸进行染色体示踪 目的3利用寡聚FISSEQ技术追踪染色体

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