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

Ohio University Athens, Athens OH

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Abstract

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.