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

使用三链体​​形成寡核苷酸作为随机分子探针进行 DNA 分析的超分辨率光学绘图

• 批准号: 10653619
• 负责人: Jixin Chen
• 金额: $ 44.81万
• 依托单位: OHIO UNIVERSITY ATHENS
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-01 至 2026-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10653619
• 关键词: Academic Research Enhancement Awards; Appalachian Region; Area; Bacteriophage lambda; Bar Codes; Base Pairing; Benchmarking; Binding; Biochemical; Bioinformatics; Biomedical Research; Biotechnology; Cell Culture Techniques; Consumption; DNA; DNA Fragmentation; DNA analysis; DNA mapping; Data; Data Analyses; Dyes; Educational process of instructing; Escherichia coli; Evaluation; Female; Fingerprint; Fluorescence; Funding; Genome; Genome Mappings; Goals; High School Student; Image; Imaging Techniques; Immobilization; Kinetics; Knowledge; Label; Light; Location; Maps; Measures; Methods; Modification; Molecular; Molecular Probes; Ohio; Oligonucleotide Probes; Oligonucleotides; Optics; Positioning Attribute; Preparation; Quality Control; Reaction; Research; Research Project Grants; Resolution; Sampling; Single-Stranded DNA; Software Tools; Stretching; Students; System; Techniques; Time; Training; Training Programs; Underrepresented Minority; Universities; Vertebral column; base; density; design; experience; fluorescence imaging; genome sequencing; graduate school; graduate student; innovation; light microscopy; method development; mid-career faculty; nanometer; next generation sequencing; novel; optical imaging; restriction enzyme; scaffold; single molecule; stem; superresolution imaging; ultra high resolution; undergraduate student; whole genome

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.

摘要 基因组光学作图是基因组测序项目的质量控制方法。PI提出了一个高- 影响和创新合作研究项目，以开发非侵入性随机分子探针， 超分辨率基因组光学作图超分辨率基因组光学作图可以显著增强 支架信息的准确性，并可以解决传统光学位置的支架 因此显著提高了DNA组装项目的质量。传统方法 在理论上限制在~300 nm（或~800碱基）分辨率，而超分辨率技术 没有理论限制。然而，有几个技术挑战，PI已经确定了一个是 侵入性荧光标记方法用于目前的光学映射方法。为了获得 在光学图谱中，必须将荧光标记物引入到靶DNA的特定位置。两 在传统的光学作图平台上，靶DNA要么被限制酶完全切割，要么被单链切割 通过一个缺口标记系统。因此，DNA在具有高标记密度的位置处断裂，并且片段 物理上的损失使得在这些位置的超分辨率成像是不可能的。PI建议开发 非侵入性荧光标记探针作为这个问题的解决方案。该提案的目标是：（1） 将超分辨率光学显微镜应用于基因组光学绘图;（2）开发非侵入性荧光 具有可调标记密度的标记探针;（3）实现荧光探针的随机荧光开-关; 用于超分辨率成像的荧光标记;（4）加强俄亥俄州大学的研究，并激励 对本科生、研究生和高中生进行研究教育。该项目引入了 生物化学和生物医学研究经验，以代表性不足的少数民族和女学生， 阿巴拉契亚地区，否则谁会缺乏这样的机会。学生将体验技术和 涉及细胞培养、DNA提取、纯化和修饰、DNA固定 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， 基因组光学作图、下一代测序（NGS）以及各种数据分析和生物信息学 方法.