Topoisomerase I Drug Screen Kit
(Catalog # K003)
This assay kit contains reagents for routine detection of
type I topoisomerase activity for drug screening
purpose. Human topoisomerase I (topoI) is the sole
intracellular target of camptothecin and other
"topo I poisons", some of which are among the most
promising anticancer drugs ever identified. The
Topo I Drug screen kit has great immense value
since it will enable the rational screening of new
anticancer compounds that might one day be used to
relieve the suffering of people diagnosed with
cancer. DNA marker is included to allow clear
detection of the enzyme. The assay is specific for
topoisomerase I. Human topoisomerase I,
active (Cat# P005) is strongly recommended to be
used along with this kit.
- Studying the effects of supercoiling on transcription in vitro.
- Studying chromatin reconstitution in vitro.
- Determining the degree of supercoiling of naturally occurring DNA.
- Detecting mutant plasmids that differ in length by only one basepair.
- Increasing restriction endonuclease digestion of resistant
DNA substrates by unwinding the DNA coils to
expose restriction sites.
- Anticancer drug screening.
Quality Control Tests:
- A test for
nuclease contamination is carried out by assaying
for the formation of linear KDNA and linear
plasmid DNA. Incubation with KDNA or supercoiled
pGEM 5Z DNA (4 hrs. at 37oC in the presence of 10
mM MgCl2) is performed. Linear DNA or
breakdown products are not generated under these
- A check for cross contamination with Topo II is negative.
There must be no decatenation of KDNA in topo II
- Supercoiled plasmid substrate
DNA control (cat# D001, 100ul pLAE1
DNA, 0.2 ug/ul, good
for 100 standard assays).
- Relaxed plasmid DNA marker
(cat# D002, 100 ul pre-relaxed pLAE1 DNA by Topo I at 0.04 ug/ul in
gel loading buffer, good for 20 standard
- 5X Topo I reaction buffer (cat# B001, 500 ul, good for 100 standard
- Topo I Stop buffer/gel loading dye (cat# B002, 500 ul, good for 100 standard assays).
- Control inhibitor (cat# B003, Camptothecin; lyophilized).
assays are recommended to carry out in a final
volume of 10 ul with 5 unit of Topo I, active
(Cat# P005) in Topo I reaction buffer (40 mM
Tris-Cl, pH 7.5, 100 mM NaCl or KCl, 10mM
MgCl2, 0.5 mM EDTA, 30 ug/ml BSA).
Assays: Supercoiled plasmid DNA (Cat# D001) is
used at 0.2 ug/reaction. Reactions are terminated
with 5 ul (in 10 ul reaction volume) of stop
buffer (5% sarkosyl, 0.0025% bromophenol blue, 25%
glycerol). Reaction products can then be analyzed
on a 0.8% native agarose gel.
- Load 5 ul (0.2 ug) Relaxed plasmid DNA marker (Cat# D002)
per lane as control.
This kit is shipped at ambient
temperature and should be stored at
consideration: FOR RESEARCH USE
Reference: Wang, J.C. (1996) Annu. Rev. Biochem. 65: 635-692.
A unit of topo I incubated with
0.2 ug pGEM 5Z DNA in 30 min at 37oC.