Xiong Kan, Blainey Paul C
Broad Institute, Massachusetts Institute of Technology and Harvard Medical School; Dept. of Biological Engineering, Massachusetts Institute of Technology.
Broad Institute, Massachusetts Institute of Technology and Harvard Medical School; Dept. of Biological Engineering, Massachusetts Institute of Technology;
J Vis Exp. 2017 Oct 1(128):55923. doi: 10.3791/55923.
We describe a simple, robust and high throughput single molecule flow-stretching assay for studying 1D diffusion of molecules along DNA. In this assay, glass coverslips are functionalized in a one-step reaction with silane-PEG-biotin. Flow cells are constructed by sandwiching an adhesive tape with pre-cut channels between a functionalized coverslip and a PDMS slab containing inlet and outlet holes. Multiple channels are integrated into one flow cell and the flow of reagents into each channel can be fully automated, which significantly increases the assay throughput and reduces hands-on time per assay. Inside each channel, biotin-λ-DNAs are immobilized on the surface and a laminar flow is applied to flow-stretch the DNAs. The DNA molecules are stretched to >80% of their contour length and serve as spatially extended templates for studying the binding and transport activity of fluorescently labeled molecules. The trajectories of single molecules are tracked by time-lapse Total Internal Reflection Fluorescence (TIRF) imaging. Raw images are analyzed using streamlined custom single particle tracking software to automatically identify trajectories of single molecules diffusing along DNA and estimate their 1D diffusion constants.
我们描述了一种简单、稳健且高通量的单分子流动拉伸测定法，用于研究分子沿DNA的一维扩散。在该测定法中，玻璃盖玻片通过与硅烷-聚乙二醇-生物素的一步反应进行功能化。流动池是通过将带有预切割通道的胶带夹在功能化盖玻片和含有进、出口孔的聚二甲基硅氧烷（PDMS）平板之间构建而成。多个通道集成在一个流动池中，试剂流入每个通道的过程可以完全自动化，这显著提高了测定通量并减少了每次测定的实际操作时间。在每个通道内，生物素化的λ-DNA固定在表面，并施加层流以流动拉伸DNA。DNA分子被拉伸至其轮廓长度的80%以上，并作为空间扩展模板用于研究荧光标记分子的结合和运输活性。通过延时全内反射荧光（TIRF）成像跟踪单分子的轨迹。使用简化的定制单粒子跟踪软件分析原始图像，以自动识别沿DNA扩散的单分子轨迹并估计其一维扩散常数。
