Topoisomer gel retardation: detection of anti-Z-DNA antibodies bound to Z-DNA within supercoiled DNA minicircles.
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.
Nordheim
A
A
Zentrum für Molekulare Biologie (ZMBH), Universität Heidelberg, FRG.
Meese
K
K
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D001483:Base Sequence; D002850:Chromatography, Gel; D004278:DNA, Superhelical / Q000276:immunology; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010957:Plasmids
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D004278:DNA, Superhelical