Pfannschmidt C, Langowski J
Division Biophysics of Macromolecules, German Cancer Research Center, Heidelberg, Germany.
J Mol Biol. 1998 Jan 30;275(4):601-11. doi: 10.1006/jmbi.1997.1476.
For determining the position of a defined site in a superhelical DNA we have developed a method for introducing a covalent biotin label at a specific sequence while preserving the superhelicity. This is done by first introducing a specific nick, labeling the DNA by limited nick translation and sealing the nick with ligase. The superhelicity is controlled by including ethidium in the ligation reaction. Using scanning force of microscopy on DNAs labeled by this method, we have then compared the position of streptavidin markers at a specific site relative to the end loop of the superhelix. We found that in DNAs with permanently curved inserts the label is located preferentially at a defined distance from the end loop, while in controls without curved inserts the label position was random. This indicates that curves are located in or near the end loops in a superhelix.
为了确定超螺旋DNA中特定位点的位置，我们开发了一种方法，即在保留超螺旋结构的同时，在特定序列处引入共价生物素标记。具体做法是先引入一个特定的切口，通过有限的切口平移对DNA进行标记，然后用连接酶封闭切口。通过在连接反应中加入溴化乙锭来控制超螺旋结构。利用扫描力显微镜观察用这种方法标记的DNA，我们比较了链霉亲和素标记在特定位点相对于超螺旋末端环的位置。我们发现，在带有永久性弯曲插入片段的DNA中，标记优先位于距末端环特定距离处，而在没有弯曲插入片段的对照DNA中，标记位置是随机的。这表明弯曲结构位于超螺旋的末端环内或其附近。
