Yokota H, Fung K, Trask B J, van den Engh G, Sarikaya M, Aebersold R
Department of Molecular Biotechnology and Material Sciences & Engineering, University of Washington, Seattle 98195, USA.
Anal Chem. 1999 May 1;71(9):1663-7. doi: 10.1021/ac981370x.
We have developed a fluorescence-based method for mapping single or multiple protein-binding sites on straightened, large-size DNA molecules (> 5 kbp). In the described method, protein-DNA complexes were straightened and immobilized on a flat surface using surface tension. A fraction of the immobilized complexes displayed a sharp DNA bend with two DNA segments extending from the apex. The presence of DNA-binding proteins at the apex was verified by atomic force microscopy. The position of protein binding relative to the ends of the DNA molecule was determined by measuring the length of two DNA segments using fluorescence microscopy. We demonstrate the potential of the fluorescence-based method to localize protein-binding sites on the DNA template and to evaluate relative binding affinity. The proposed protein-binding-site mapping technique is simple and easy to perform. Practical applications include screening for DNA-binding proteins and the localization of protein-binding sites on large segments of DNA.
我们开发了一种基于荧光的方法，用于在拉直的大尺寸DNA分子（> 5 kbp）上绘制单个或多个蛋白质结合位点。在所描述的方法中，蛋白质-DNA复合物通过表面张力被拉直并固定在平坦表面上。一部分固定的复合物显示出尖锐的DNA弯曲，有两个DNA片段从顶点延伸出来。通过原子力显微镜验证了顶点处存在DNA结合蛋白。通过使用荧光显微镜测量两个DNA片段的长度，确定了蛋白质结合相对于DNA分子末端的位置。我们展示了基于荧光的方法在定位DNA模板上的蛋白质结合位点以及评估相对结合亲和力方面的潜力。所提出的蛋白质结合位点映射技术简单且易于操作。实际应用包括筛选DNA结合蛋白以及在大片段DNA上定位蛋白质结合位点。
