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1、METHODSINMOLECULARBIOLOGYMETHODSINMOLECULARBIOLOGYTMVolume282ApoptosisApoptosisMethodsMethodsandProtocolsandProtocolsEditedbyEditedbyHughJ.M.BradyHughJ.M.BradyMeasurementofApoptosisbyDNAFragmentation11MeasurementofApoptosisbyDNAFragmentationDemetriusMatassov,TerriKagan,JulieL
2、eblanc,MariannaSikorska,andZahraZakeriSummaryClassicalapoptoticcelldeathcanbedefinedbycertainmorphologicalandbio-chemicalcharacteristicsthatdistinguishitfromotherformsofcelldeath.Onesuchfeature,whichisahallmarkofapoptosis,isDNAfragmentation.Indyingcells,DNAiscleavedbyanendonu
3、cleasethatfragmentsthechromatinintonucleosomalunits,whicharemultiplesofabout180-bpoligomersandappearasaDNAladderwhenrunonanagarosegel.Here,wepresentcommonlyusedmethodssuchasconventionalagarosegelelectrophoresistoanalyzefragmentednucleiincells.Thevariousmethodsusedaredependent
4、ontheextentoffragmentationortheamountoffragmentednucleiinasample.DeterminingwhetheracellexhibitsDNAfrag-mentationcanprovideinformationaboutthetypeofcelldeathoccurringandthepathwaysactivatedinthedyingcell.KeyWords:Apoptosis;DNAfragmentation;electrophoresis;pulse-field;agaroseg
5、el.1.IntroductionThemethodsdescribedinthischapterdetectdamagedDNAincellsthathavefragmentedtheirgenomeduringprogrammedcelldeathorapoptosis.Apoptosisisanactiveregulatorycellularresponsetocertainstimuli,suchaslossoftrophicfactorsupport(serumdeprivation)orexposuretocytotoxicagent
6、s(chemotherapeuticFrom:MethodsinMolecularBiology,vol.282:ApoptosisMethodsandProtocolsEditedby:H.J.M.Brady©HumanaPressInc.,Totowa,NJ12Matassovetal.drugs),thatresultsinasomewhatstereotypedcelldeath(1–4),includingmembraneblebbing,nuclearcondensation,andthefor-mationofapoptoticbo
7、dies.Abiochemicalhallmarkofapoptosisisthecleavageofchromatinintosmallfragments,includingoligo-nucleosomes,thatwhenseeninelectrophoresedgelsaredescribedas“DNAladders”(5,6).DNAfragmentationwasfirstdocumentedbyWilliamsonin1970whenheobserveddiscreteoligomericfrag-mentsoccurringdu
8、ringcelldeathinprimaryneonatallivercultures(7).Afterthatdiscovery,Wi