Nordheim A, Meese K
Zentrum für Molekulare Biologie (ZMBH), Universität Heidelberg, FRG.
Nucleic Acids Res. 1988 Jan 11;16(1):21-37. doi: 10.1093/nar/16.1.21.
Small DNA fragments of approximately 350 bp in length, either with or without d(CG)n tracts, are ligated into underwound DNA minicircles to generate topoisomeric rings with different topological linking numbers, Lk. These minicircles, differing by an Lk of one, can be separated by acrylamide gel electrophoresis. Furthermore, electrophoresis can be used to reveal DNA double helix conformational changes that are induced by supercoiling, such as left-handed Z-DNA. When anti-Z-DNA antibodies are added to such minicircles, their binding leads to a selective retardation of the electrophoretic migration of the Z-DNA containing circles. This effect is not seen with relaxed minicircles and those with insufficient torsional stress to induce a conformational transition. Thus the technique of 'topoisomer gel retardation' presents a very sensitive assay for the identification of proteins that selectively bind to DNA conformations stabilized by negative DNA supercoiling.
长度约为350 bp的小DNA片段，无论有无d(CG)n序列，都被连接到解旋的DNA小环中，以产生具有不同拓扑连接数Lk的拓扑异构体环。这些小环比值相差1个Lk，可以通过丙烯酰胺凝胶电泳分离。此外，电泳可用于揭示由超螺旋诱导的DNA双螺旋构象变化，如左手Z-DNA。当将抗Z-DNA抗体添加到此类小环中时，它们的结合会导致含Z-DNA环的电泳迁移选择性延迟。对于松弛的小环和扭转应力不足以诱导构象转变的小环，不会出现这种效应。因此，“拓扑异构体凝胶阻滞”技术提供了一种非常灵敏的检测方法，用于鉴定选择性结合由负DNA超螺旋稳定的DNA构象的蛋白质。
