
LinearKinetoplastDNAMarkerDescription
AssaysemployingcrudeextractsfortopoIIactivitybaseduponrelaxationofsupercoiledDNAcanbecomplicatedduetothepresenceoftopoIinpartiallypurifiedfractions. Additionalcomplicationsarisewithcontaminatingnucleaseactivity(duetoMg++)whichdegradeornickthesupercoiledsubstrate. TheseproblemscanbeavoidedbyusingacatenatedDNAsubstratepreparedfromthekinetoplastoftheinsecttrypanosomeCrithidiafasciculata. KDNAisanaggregateofinterlockedDNAminicircles(mostly2.5kb)thatformextremelylargenetworksofhighmolecularweight. Asaresult,thesenetworksfailtoenteranagarosegel.UponincubationwithtopoII,whichengagesDNAinadoublestrandedbreakingandreunioncycle,minicircularDNAsareeffectivelyreleased(decatenated). Thedecatenatedminicirclesmoverapidlyintothegelowingtotheirsmallsize. ThisreactionwillnotoccurwithtopoisomeraseI.TopoGENhasmodifiedtheoriginalgelsystemdescribedbyMilleretal.(seebelow)toallowadditionaldiscriminationofactivitiesusingkDNA. Theproductsofthereactioncanvaryasfollows:
QualityControlTests:
- ForcatenatedKDNAsubstrate,atleast90%oftheDNAwillberetainedinthewellofa1%agarosegel.
- DecatenationofeachbatchofkDNAistestedwithpurifiedtopoisomeraseII.
References:
- Milleretal.,J.Biol.Chem.256:9334-9339(1981)
- Mulleretal.,Biochem.27:8369-8379,1988
- Mulleretal.,Nuc.Acid.Res.17:9499,1989