The Topo I Drug Kit introduces transient nicks in DNA at specific sites. Detection of these transient DNA nicks requires trapping the enzyme on DNA in a nicked intermediate complex using protein denaturants. The resulting covalent DNA/topo I complexes contain nicked open circular DNA which can be detected by agarose gel electrophoresis (with ethidium bromide). Trapping nicked intermediates is relatively inefficient; however, inhibitors, such as the natural product camptothecin, stabilize the intermediate and lead to an increase in the nicked DNA product. This forms the basis for a mechanistic drug screening assay designed to allow detection of agents that affect topoisomerase I by stabilizing the cleaved intermediate complex. The TopoGEN Topo I Drug Screening Kit is designed to allow the investigator to quickly identify novel inhibitors of Human Top1 (included with kits denoted -A in catalog numbers). The kit will allow detection of novel compounds that either stabilize the nicked intermediate or otherwise inhibit catalytic activity of topoisomerase I.
Topoisomerase I Drug Screening Kit Contents (representative for 100 assay kit size):
-100 Units Human Top1 Enzyme (available with catalog number TG1018-1A only)
-Supercoiled pHOT-1 DNA, 25 µg
-Relaxed and nicked plasmid DNA markers
-10X Topoisomerase I assay/cleavage buffer, 300 ul
-Sodium dodecyl sulfate (SDS) termination buffer (10%)
-10X gel loading dye
-Control inhibitor (Camptothecin; lyophilized)
-Detailed instruction manual
The Topoisomerase I Drug Screening Kit is designed to work with human topo I available from TopoGEN. Since cleavage assays require more enzyme than catalytic assays, TopoGEN sells the enzyme in larger lots which takes this difference into account.
Use of the Topoisomerase I Drug Kit
Reactions indicated above each lane were carried out using 5 units of Top1. The reactions were incubated for 30 min at 37 C and stopped with SDS. After digestion with proteinase K (50 ug/ml, 60 min at 37 C), samples were loaded onto a 1% agarose gel. Under these conditions, with excess topo I and camptothecin, significant conversion of supercoiled (form I) DNA to open circular (OC DNA) was observed. It is important to recognize that cleavage assays will consume more enzyme than catalytic (relaxation type) assays. This is because each cleavage event consumes one enzyme molecule; i.e., the reaction requires stoichiometic amounts of topo I.
This product is shipped at ambient temperature or dry ice if included with enzyme. Store enzyme at -20°C. Store remaining kit contents at 4°C upon receipt.