Study On Three-dimensional Nano-resolution Visualization Of Specific Nonrepetitive DNA In Human Genome By STORM

Direct visualization of any specific DNA within a single human cell is essential for understanding the structure and function of the human genome. However, the characteristic scale of these DNA image is in the nanometer range, which is far beyond the resolution limit of conventional optical microscope. In recent years, with the development of far-field super-resolution fluorescence microscopy, there are several microscopy approaches that break the diffraction-limit, achieving lateral(x, y) spatial resolution of 10-20 nm and 20-50 nm in the axial(z), hence becoming powerful research tools for DNA research. Currently, the researchers have obtained some high resolution images of some repetitive or non-specific chromosomal DNA, but it still remains challenging to visualize a region of specific non-repetitive DNA in nuclear genome. For example, how to efficiently and specifically la bel and image nuclear non-repetitive DNA sequences at nanometer resolution. To solve this problem, we carry out the research based on the method of three-dimensional stochastic optical reconstruction microscopy(3D-STORM), the main content is as follows:1. Described the basic theory and some technology problems of STORM, introduced the home-built 3D STORM microscopy in our laboratory in details.2. Optimized the existing super-resolution fluorescence imaging system so as to visualize specific non-repetitive DNA in nuclear genome: designed and finished experiment to improve imaging buffer; introduced 405 nm laser as a activation light for fluorescent molecule. Experimentally calibrated the ability of the system to image fluorescent molecules in super-resolution.3. Used a novel FISH probe-- Alexa 647-linked molecular beacon(MB), which could specifically label the DNA of interest, and checked its quality. Modified a lentiviral p LL3.7 vector and inserted it into nuclear genome of human SK-N-SH neuroblastmoa cells as target DNA via transfection. Sorted out the transfected SK-N-SH cells by flow cytometry, confirmed the integration and visualized it.4. Finished the experiment of visualizing the specific non-repetitive DNA using the optimized existing super-resolution fluorescence imaging system, collected data during the experiment, analyzed those collected data for reconstruction. Finally, got the 3D structures image of the non-repetitive target DNA at nanometer resolution.