This kit is designed to investigate whether a compound intercalates into the DNA double-helix, or binds in the groove, leading to unwinding of the DNA. This is a characteristic of a number of inhibitors of DNA-modifying enzymes such as the topoisomerase inhibitor m-amsacrine.
Intercalators contain planar, normally polycyclic, aromatic structures which can insert between the bases of the double-helical DNA molecule. Compounds which are able to intercalate into DNA or bind in the groove can lead to local unwinding of the DNA leading to a decrease in the twist of the DNA. If a DNA molecule is nicked and rejoined (for example by a topoisomerase) in the presence of such a compound, then the result is relaxed, underwound DNA; on removal of the enzyme and compound this converts to supercoiled DNA. The supercoiled DNA formed in these conditions is indicative of an intercalator.
This is the basis of the DNA unwinding assay. Supercoiled (or relaxed) plasmid DNA is incubated briefly with the test compound prior to relaxation by the wheat germ topo I. The enzyme and drug are then removed by extraction and the plasmid analysed by gel electrophoresis. Supercoiled topoiosmers indicate the compound is an intercalator or groove binder. If the compound is not an intercalator then the product will be relaxed plasmid.
The assay should be checked by performing a control reaction using relaxed plasmid to show that the compound is not simply acting as an inhibitor of the topo I.
The kit (product code DUKSR001) contains all you need to do the assay including:-
Supercoiled pBR322 (50 μg), relaxed pBR322 (25 μg), wheat germ topo I (250 U), Assay Buffer (1 ml) and Dilution Buffer (1 ml).
We also supply kits which contain topo I and buffers but only:-
(i) the relaxed form of pBR322 (product code DUKR002). This will show if a compound is an intercalator but not if it also inhibits the topo I (i.e. false-negatives are possible)
(ii) the supercoiled form of pBR322 (product code DUKS003). This will show if a compound is an intercalator but the same result will be given if it is an inhibitor of topo I (i.e. false-positives are possible)
This is illustrated in the scheme below: