‘Gel-Stacks’ gently confine or reversibly immobilize arrays of single DNA molecules for manipulation and study

Large DNA molecules (>20 kb) are difficult analytes prone to breakage during serial manipulations and cannot be ‘rescued’ as full-length amplicons. Accordingly, to present, modify and analyze arrays of large, single DNA molecules, we created an easily realizable approach offering gentle confinement conditions or immobilization via spermidine condensation for controlled delivery of reagents that support live imaging by epifluorescence microscopy termed ‘Gel-Stacks.’ Molecules are locally confined between two hydrogel surfaces without covalent tethering to support time-lapse imaging and multistep workflows that accommodate large DNA molecules. With a thin polyacrylamide gel layer covalently bound to a glass surface as the base and swappable, reagent-infused, agarose slabs on top, DNA molecules are stably presented for imaging during reagent delivery by passive diffusion. Gel-Stacks technology provides multiple non-covalent molecular presentation modes, coupled with an unusually facile reagent delivery system designed for large-scale analytes, enhancing live imaging and manipulation. Enhanced further by modeling and software, Gel-Stacks technology becomes adaptable to a broad range of experimental applications.

长度大于20千碱基对（kb）的大型DNA分子属于较难处理的分析物，在连续实验操作过程中极易发生断裂，且无法被完整挽救为全长扩增子。为此，为实现大型单个DNA分子阵列的呈现、修饰与分析，我们开发了一种易于实现的技术方案：通过亚精胺（spermidine）缩合提供温和的受限环境或固定方式，实现试剂的可控递送，可支持落射荧光显微镜（epifluorescence microscopy）下的活细胞成像，该技术被命名为"凝胶堆叠（Gel-Stacks）"。该技术将分子局部限制于两层水凝胶表面之间，无需共价锚定，可支持延时成像以及适配大型DNA分子的多步实验流程。其基底为共价结合于玻璃表面的超薄聚丙烯酰胺凝胶（polyacrylamide gel）层，上方放置可更换、浸润了试剂的琼脂糖凝胶块（agarose slab），DNA分子可通过被动扩散完成试剂递送，并在该过程中被稳定呈现以用于成像。凝胶堆叠技术提供多种非共价式分子呈现模式，并搭配专为大规模分析物设计的极为简便的试剂递送系统，可优化活细胞成像与分子操作流程。通过建模与软件进一步优化后，凝胶堆叠技术可适配广泛的实验应用场景。