Hu X, Aston C, Schwartz D C
Laboratory for Biomolecular Imaging, New York University, New York, New York, 10003, USA.
Biochem Biophys Res Commun. 1999 Jan 19;254(2):466-73. doi: 10.1006/bbrc.1998.9971.
Single molecule approaches to the characterization of biochemical systems offer an intrinsically simple and direct approach to address difficult, previously unyielding problems. Optically based approaches have recently been used to construct high resolution, ordered restriction maps from a variety of clone types. Advancements in surface technologies have enabled the reliable elongation and fixation of large DNA molecules onto specially derivatized substrates with retention of biochemical accessibility. In this study, the addition of fluorescently labeled nucleotides to surface-mounted DNA molecules by the action of DNA polymerase I is investigated using fluorescence microscopy to image individual template molecules. Molecules undergoing nick translation and containing only a few fluorochromes are readily imaged. These novel results suggest that surface-bound molecules may serve as a substrate for a broad range of enzymatic actions, and may offer new routes to analysis when coupled to advanced imaging techniques.
用于生物化学系统表征的单分子方法提供了一种本质上简单直接的方法来解决以前难以解决的棘手问题。基于光学的方法最近已被用于从各种克隆类型构建高分辨率、有序的限制性图谱。表面技术的进步使得能够将大的DNA分子可靠地延伸并固定到经过特殊衍生的底物上，同时保留生化可及性。在本研究中，利用荧光显微镜对单个模板分子进行成像，研究了通过DNA聚合酶I的作用将荧光标记的核苷酸添加到表面固定的DNA分子上的情况。进行切口平移且仅含有少量荧光染料的分子很容易成像。这些新结果表明，表面结合的分子可能作为广泛酶促作用的底物，并且当与先进的成像技术结合时可能提供新的分析途径。
