KDNA Complications

Complications when using kDNA as a substrate for topo II assays:

The most serious complications arise when there are interfering proteins or substances in the extract being assayed. Crude, cell free extracts may contain excessive amounts of DNA binding proteins or positively charged proteins that stick to the DNA and inhibit enzyme access. Also, nuclease contaminants may degrade or nick the KDNA substrate and therefore obscure the results. A good way to deal with this problem includes cleaning up crude extracts by ammonium sulfate precipitation followed by column chromatography. Also, by diluting extracts and/or adding a tRNA carrier (to compete basic proteins), one can sometimes minimize such problems.

Additionally, it is extremely important to run appropriate controls and markers when using kDNA. You , MUST be able to unambiguously identify kDNA decatenation products (nicked monomers, circular monomers (relaxed vs. supercoiled), linear DNAs).

Note also that some lots of KDNA may have a bit of chromosomal DNA from Crithidia genome. This genomic DNA will migrate above any decat. products and should NOT interfere with your assays.

We offer custom screening on a contract basis. Let us test your compounds for topo targeting activity.