Super-Resolution Optical Mapping for DNA Analysis Using Triplex-Forming Oligonucleotides as Stochastic Molecular Probes

摘要 基因组光学作图是基因组测序项目的一种质量控制方法。PI提出了一个高- 影响和创新合作研究项目，开发非侵入性随机分子探针 超分辨率基因组光学测绘。超分辨率基因组光学作图可以显著提高 脚手架信息的准确性，并可以在传统光学位置解析脚手架 作图不能，因此显著提高DNA组装项目的质量。传统的方法 理论上限制在~300纳米(或~800个碱基)的分辨率，而超分辨率技术 没有理论上的限制。然而，存在几个技术挑战，PI已确定其中一个是 目前光学标记法中采用的是侵入性荧光标记法。为了获得一个 在光学图谱中，荧光标签必须被引入到特定位置的目标DNA上。在这两个中 传统的光学作图平台，目标DNA要么被限制性内切酶完全切割，要么被单链切割 通过尼克标签系统。因此，DNA在具有高标记密度的位置断裂，并且片段 物理上的损失，使得在这些地点进行超分辨率成像是不可能的。PPI建议发展 非侵入性荧光标记探针作为这一问题的解决方案。该提案的目标是：(1) 将超分辨光学显微镜应用于基因组光学作图；(2)研制非侵入性荧光 标记密度可调的标记探针；(3)实现随机荧光开关 用于超分辨率成像的荧光标记；(4)加强俄亥俄大学的研究，并激励 对本科生、研究生和高中生进行研究教育。这个项目已经引入了 为未被充分代表的少数民族和女性学生提供生物化学和生物医学研究经验 阿巴拉契亚地区，否则他们将缺乏这样的机会。学生们将体验技术和 在细胞培养、DNA提取、纯化和修饰、DNA固定化、 探针设计，探针合成，DNA标记，传统荧光成像，超分辨率光学成像， 基因组光学测绘、下一代测序(NGS)以及各种数据分析和生物信息学 方法：研究方法。

Abstract Genome optical mapping is a quality control method for a genome sequencing project. The PI proposes a high- impact and innovative collaborative research project to develop non-invasive stochastic molecular probes for super-resolution genome optical mapping. Super-resolution genome optical mapping can significantly enhance the accuracy of the scaffolding information and can resolve the scaffolds at locations where traditional optical mapping cannot, thus significantly increasing the quality of a DNA assembly project. The traditional methods have a theoretical limitation at ~300 nm (or ~800 bases) resolution while the super-resolution techniques have no theoretical limit. However, there are several technical challenges and the PI has identified one to be the invasive fluorescent labeling methods used in the current optical mapping methods. In order to obtain an optical map, fluorescence tags have to be introduced to the target DNA at specific locations. In the two traditional optical mapping platforms, the target DNA is either fully cut by a restriction enzyme or one-strand cut by a nick labeling system. As such, the DNA breaks at locations with high labeling density, and the fragments are physically lost making super-resolution imaging at these locations impossible. The PI proposes to develop non-invasive fluorescence labeling probes as a solution to this problem. The goals of the proposal are: (1) apply super-resolution light microscopy in genome optical mapping; (2) develop non-invasive fluorescent labeling probes that have tunable labeling densities; (3) achieve stochastic fluorescent ON-OFF of the fluorescent tags for the super-resolution imaging; (4) enhance the research at Ohio University, and motivate and educate the undergraduate, graduate, and high school students in research. This project has introduced biochemical and biomedical research experience to underrepresented minority and female students in the Appalachian area, who would otherwise lack such opportunities. The students will experience techniques and knowledge that span widely in cell culture, DNA extraction, purification, and modification, DNA immobilization, probe design, probe synthesis, DNA labeling, traditional fluorescent imaging, super-resolution optical imaging, genome optical mapping, next-generation sequencing (NGS), and a variety of data analysis and bioinformatics methods.