‘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.

大于20千碱基对（kb）的大型DNA分子属于难以操控的分析物，在连续操作过程中极易发生断裂，且无法获得全长扩增子。为此，为了呈现、修饰并分析大型单链DNA分子阵列，我们开发了一种易于实现的方法，该方法可提供温和的约束条件，或通过亚精胺缩合（spermidine condensation）实现固定化，以支持适配落射荧光显微镜（epifluorescence microscopy）活细胞成像的试剂可控递送，此方法被命名为“凝胶堆叠（Gel-Stacks）”。该方法使DNA分子被局部约束于两层水凝胶表面之间，且无需共价锚定，可支持延时成像及适配大型DNA分子的多步实验流程。该体系以共价结合于玻璃表面的薄聚丙烯酰胺凝胶层作为基底，上方辅以可更换、浸润了试剂的琼脂糖平板，DNA分子可通过被动扩散稳定呈现，以完成试剂递送过程中的成像。凝胶堆叠技术提供了多种非共价分子展示模式，并搭配专为大规模分析物设计的极为便捷的试剂递送系统，可优化活细胞成像与操作流程。结合建模与软件工具进一步优化后，凝胶堆叠技术可适配广泛的实验应用场景。