TheGyraseAssayKitProductDescription
TheKitisdesignedtoallowquickandspecificdetectionofDNAgyrase.ThiskitfacilitatesthepurificationandcharacterizationoftypeIItopoisomeraseenzymes(DNAGyrase)andcontainsallreagentsnecessaryforroutineassaysoftypeIIenzymesthateitherhaveordonothavetheABIlitytosupercoil.

TheGyraseAssayisBasedUponaTwoStepProcess:

1. DecatentationofkinetoplastDNA(kDNA)
2. SupercoilingoftheresultingdecatenatedmonomerkDNAspecies

ReactionproductsareresolvedusinganovelgelsystemdevelopedbyTopoGENthatallowsextremelyrapidandunambiguousdetectionofgyraseactivity. Theappropriatebuffers,DNAsubstratesandDNAMarkersareincluded. Purifiedgyraseisnotincludedinthiskit(TG2000G);however,decatenatedandlinearDNAmarkersareincludedtoallowclearandfacileidentificationofproductsintheextractsorfractionsdefinedbytheuser(seelinkabove). ThekDNAassaywilldetectpoisonsthatstimulateDNAcleavagebygyraseaswellasagentsthatsimplyinhibitcatalyticactivity. TheadvantageofakDNAbasedgyraseassayisthatitisfastandeasytoallowactivityguidedpurificationofinhibitorsfromcrudemixturesorextracts.

GyraseAssayKitContents:

-KinetoplastDNA[kDNA]sufficientfor100assays
-MarkerlinearkDNA
-MarkerdecatenatedkDNA(topoIItreated)
-10XGyraseassaybuffer
-Gyrasestopandloadbuffer
-Detailedinstructionmanual.

ApplicationNotesforDNAGyraseAssayKit:
ThedegreeofdecatenationofKDNAbytopoisomeraseIIorDNAgyraseisproportionaltotheamountofenzymeandthelengthofincubation. Forscreeningofcolumnfractionstodetermineanactivitypeak,itisbesttouseashort(5minute)incubationperiodsothatonlythemostactivefractionswillcompletelydecatenatethekDNA. Forscreeningofcolumnfractionsorotherassaysinwhichmanyreactionswillbeconducted,itisbesttosetupamasterreactionmixturecontainingallreagentsexceptthetestfraction,thenaliquotingthisintothereactiontubestowhichthefractionswillbeadded.

Highconcentrationsofsaltinhibittopoisomerases;therefore,testfractionsshouldnotcontributegreaterthan30mMmonovalentsalttothefinalreaction. Iftheionconcentrationexceedsthis,thenalowsaltbuffershouldbeusedtocompensate.

Itisbesttoincludeacompletereactionlackingproteinfraction,butcontainingthebufferinwhichthetestfractionisfound. IftheproteinfractionscontainagentswhichaltertheelectrophoreticmobilityofDNAitmaybenecessarytophenol/chloroformtreatand/orethanolprecipitatesamplesbeforeloADIngthegels.

InterpretationofGels:

ThedecatenatedDNAmarkerwillbeOpenCircularDNA(OC,2.5KBmonomerswithatleastonenick)andsomeClosedCircularDNA(CC)DNA. ThekDNAusuallycontainsnickedDNAmonomers;thus,bothOCandCCDNAwillbeseen. Incontrast,linearDNAshouldnotbeproducedsincethisindicatesnucleasecontamination. NotealsothatonemayusethekDNAassayforDNAgyrasetoidentifyagentsthatstabilizethenickedorcleavageintermediate(seelinkabove).

Complications:
Themostseriouscomplicationsarisewhenthereareinterferingproteinsorsubstancesintheextractbeingassayed. Crude,cellfreeextractsmaycontainexcessiveamountsofDNAbindingproteinsorpositivelychargedproteinsthatsticktotheDNAandinhibitenzymeaccess. Also,nucleasecontaminantsmaydegradeornickthekDNAsubstrateandthereforeobscuretheresults. Agoodwaytodealwiththisproblemincludescleaningupcrudeextractsbyammoniumsulfateprecipitationfollowedbycolumnchromatography. Also,bydilutingextractsand/oraddingatRNAcarrier(tocompetebasicproteins),onecansometimesminimizesuchproblems.