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QUALITATIVE ANALYSIS OF DNA FRAGMENTATION BY AGAROSE GEL ELECTROPHORESIS

蚂蚁淘  /发布时间:1545758673 /浏览量:26   

1.Introduction

    Nuclearmorphologychangescharacteristicofapoptosisappearwithinthecelltogetherwithadistinctivebiochemicalevent:theendonuclease-mediatedcleavageofnuclearDNA.Infact,formationofDNAfragmentsofoligonucleosomalsize(180-200bp)isanhallmarkofapoptosisinmanycelltypes.

    ThepresentprotocolprovidesamethodforDNAseparationoffragmentedandintactDNAfractionsandfortheiranalysisbyagarosegelelectrophoresis.InapoptoticcellsspecificDNAcleavagebecomesevidentinelectrophoresisanalysisasatypicalladderpatternduetomultipleDNAfragments.However,althoughthisprotocolissimpleandgenerallyabletoprovidegoodresults,itisonlyqualitativebecauseofitslimitationsinDNArecoveryandsolubilization.InordertoobtainacleanerDNA,othermethodsforDNApreparationarerequired(insomecasesuseofproteinaseKfordeproteinizationisrecommended).

2.Protocol
    2.1.MaterialsCellsUSPensionat1-5x106cells/mlincompleteRPMImedium(A1)TTEsolution:TEbufferpH7.4(A1)with0.2%TritonX-100(storeat4°C)NaCl5M,icecoldIsopropanol,icecoldEthanolat70%,icecoldTEbufferpH7.4(A1)LoADIngbuffer10x(A1)TBEbufferforelectrophoresis(A1)Ethidiumbromidesolution(A1)Electrophoresis-gradeagaroseDNAmolecularweightMarkersRefrigeratedcellcentrifuge(A3)Microfuge(A3)Heatingblock(A3)Gelelectrophoresisapparatus(A3)DCpowersupply(A3)UVtransIlluminator(A3)PolaroidCamera+films(A3)

    2.2.Methodology

      1.Dispense0.5mlofcellsuspension(nolessthan5x105,otherwiseDNAwillnotbedetectablebyphotographyofethidiumbromidestainedgel,andnomorethan5x106,toavoiddifficulthandlingoftoohighamountsofinsolubleDNA)intubeslabeledB(bottom).

      2.Centrifugecellsat200xgat4°Cfor10min.

      3.TransfersupernatantscarefullyinnewtubeslabeledS(supernatant).

      4.AddtothepelletintubesB0.5mlofTTEsolutionandvortexvigorously.Thisprocedureallowsthereleaseoffragmentedchromatinfromnuclei,aftercelllysis(duetothepresenceofTritonX-100intheTTEsolution)anddisruptionofthenuclearstructure(followingMg++chelationbyEDTAintheTTEsolution).

      5.ToseparatefragmentedDNAfromintactchromatin,centrifugetubesBat20,000xgfor10minat4°C.

      6.CarefullytransfersupernatantsinnewtubeslabeledT(top).

      7.AddtothesmallpelletintubesB0.5mlofTTEsolution.

      8.Addtothe0.5mlvolumepresentintubesB,SandT,0.1mlofice-cold5MNaClandvortexvigorously.TheadditionofthesaltshouldbeabletoremovehistonsfromDNA.

      9.Addtoeachtube0.7mlofice-coldisopropanolandvortexvigorously.

      10.Allowprecipitationtoproceedovernightat-20°C.Thisstepcanbeshortenedbyputtingsamplesinabathofethanol/dryicefor1hr.

      11.Afterprecipitation,recoverDNAbypelletingfor10minat20,000xgat4°C.

      12.Discardsupernatantsbyaspirationorbyrapidlyinvertingtubesandcarefullyremoveanydropsorfluidremainingadherenttothewallofthetubewithapapertowelcorner.Thiscanbeacriticalstepbecausethepelletcouldbeloosenandtransparent,hardtobeseen.

      13.Rinsethepelletsbyaddingtoeachtube0.5-0.7mlofice-cold70%ethanol.

      14.Centrifugetubesat20,000xgfor10minat4°C.

      15.Discardsupernatantsbyaspirationorbyrapidlyinvertingtubes.Carefullyremoveanydropsorfluidremainingadherenttothewallofthetubebyinvertingtubesoveranabsorbentpapertowelfor30min.Letairdrythetubesinuprightpositionforatleast3hrbeforeproceeding.

      16.DissolveDNAbyaddingtoeachtube20-50mlofTEsolutionandplacethetubesat37°Cfor1-3days.TheredissolutionofDNAmaybeacricalstep,infactitdependsontheDNAquantityandsizepresentinthesamples.Thus,thenon-fragmentedDNAcontainedintheBtubes,mayneedhighervolumesofTEandlongerincubationtimesinordertoberesuspended.

      17.MixthesamplesofDNAwithloadingbufferbyadding10xloadingbuffertoafinalconcentrationof1x.Theadditionofloadingbuffertosamplesallowstoloadgelwellsmoreeasilyandtomonitortherunofsamples.

      18.Placesamplesinaheatingblockat65°Cfor10minandimmediatelyload10-20mlofthemtoeachwellofastandard1%agarosegelcontainingethidiumbromide0.5mg/ml.AppropriateDNAmolecularweightmarkersshouldbeincluded.Ethidiumbromideisapotentialcarcinogen:wearglovesandhandlewithcare.

      19.RuntheelectrophoresisinstandardTBEbufferaftersettingthevoltagetothedesiredlevel.DuringelectrophoresisitispossIBLetomonitorthemigrationofsamplesbyfollowingthemigrationofbromophenolbluedyecontainedintheloadingbuffer.

      20.Stoptheelectrophoresiswhenthedyereachesabout3cmfromtheendofthegel.

      21.TovisualizeDNA,placethegelonaUVtransilluminatorandtakephotosofthegel.WeareyeandskinprotectionwhenUVareon.

      3.Commentary
        3.1.Backgroundinformation

        Apoptosisisaninnatemechanismofeukarioticcellsuicidewhichplaysamajorroleinmanyphysiologicalandpathologicalprocesses.Therefore,thedefinitionofcellularregulatorymechanismsandbiochemicalprocessesinvolvedinapoptosisisanimportantchallengefromboththeoreticalandappliedpointsofview.

        Duringapoptosisaseriesofreorganisationoccurinthecell:chromatincondensation,lossofcellvolumeandmembraneblebbingaresomeofthemostevidentmorphologicalchangesofapoptoticcells.Althoughthemolecularmechanismsleadingtosuchchangesarenotcompletelyknown,manyofthemseemtoproceedinparallelwithbiochemicalevents.Thisisthecase,forexample,ofchromatincondensationandnuclearenvelopbreakdown.Infact,inparallelwiththemoccursDNAfragmentation,abiochemicalhallmarkofapoptosisinthemajorityofcells.ResponsibleforDNAcleavageisbelievedtobeanendogenousCa++-andMg++-dependentendonucleaseabletobreakdoublestrandDNAatinternucleosomalsites.Therefore,apoptoticDNAcleavageresultsincharacteristicfragmentsofoligonucleosomalsize(180-200bp).Suchphenomenum,describedforthefirsttimebyWyllie(1980),canbevisualizedbyanagarosegelelectrophoresisanalysis.ThepresentprotocolprovidesamethodforqualitativedeterminationofDNAfragmentation.

        3.2.Criticalparameters

          ThemostcriticalpointofDNAelectrophoreticalanalysisisitsinABIlityofquantitativemeasurementofapoptosis.Infact,duetoproblemslinkedtotheinsolubilityoflargeDNAandthustoitsfinalagarosegelanalysis,themethodisstrictlyqualitative.

          Moreover,thismethodisnotrecommendedwhendifferentbehaviourinDNAfragmentationfollowingapoptoticstimuliisdescribed.Infact,insomecelltypeswhererandomdouble-strandedorraresingle-strandedDNAfragmentationoccur,itcannotbedetectedbyagarosegelelectrophoresisassay.

          Sometimes,inparticularwithcellsobtainedfromexvivocultures(e.g.thymocytesandlymphocytes),anhighbackgroundofspontaneousDNAfragmentationcouldbeobserved.

          3.3.Troubleshooting

            Solubilizationofchromosome-lengthDNAcollectedintubesBisgenerallydifficult.IncreaseofTEvolumesandextensionofincubationtimemaybeneededfortheredissolutionofDNAfollowingprecipitation.Theuseoflimitednumberofcells(lessthan5x106)willbehelpfultolimitthisproblem.However,sincethemethodisexclusivelyqualitative,theanalysisoffragmentedDNApresentintubesTisthemaininterestoftheassay.

          3.4.Anticipatedresults

            TheassayofDNAagarosegelelectrophoresisprovidesgoodresultsforthedefinitionofcellapoptosis.AtypicalladderpatternofDNAfragmentationshouldbeobservedinmostapoptoticcells.
          3.5.Timeconsiderations
            PreparationofDNAforagarosegelelectrophoresisanalysisdependsontheDNAsizeofsamples:generallyfrom2to7daysarerequired.Additional4-8hrareneededforperformingelectrophoresis.
          3.6.Keyreferences1.Wyllie,A.H.1980.Glucocorticoid-inducedthymocyteapoptosisisassociatedwithendogenousendonucleaseactivity.Nature284:555.

          2.Duke,R.C.,andCohen,J.J.1986.Endogenousendonuclease-inducedDNAfragmentation:anearlyeventincell-mediatedcytolisis.Proc.Natl.Acad.Sci.U.S.A.80:6361.

          3.Arends,M.J.,Morris,R.J.,andWyillie,A.H.1990.Apoptosis.Theroleoftheendonuclease.Am.J.Pathol.136:593.

          4.Bortner,C.D.,Oldenburg,N.B.E.,andCidlowski,J.A.1995.TheroleofDNAfragmentationinapoptosis.TrendsCellBiol.5:21.

          5.Sellins,K.S.,andCohen,J.J.1991.CytotoxicTlymphocytesinducedifferenttypesofDNAdamageintargetcellsofdifferentorigin.J.Immunol.147:795.

          Appendix1(A1):Stocksolutions

          Appendix2(A2):Reagents

          Solution
          Preparation
          Storage
          Complete

          RPMImedium

          		RPMI-1640mediumsupplementedwith5%heat-inactivatedfetalcalfserum(FCS),2mML-glutamine,25mMHEPESbuffer,50µg/mlgentamicinsulfate.L-glutamineislabile,thusitdoesnotlastat4°Cformorethanoneday.
          4°C
          TEbuffer10mMTris.ClpH7.4(preparebydilutingstocksolution),1mMEDTA.
          RT
          Tris.Clstocksolution(1M)Dissolve121gTrisbasein800mlH2O,adjusttodesiredpHwithconcentratedHCl,mixandaddH2Oto1liter.

          CAUTION:AdjustpHoftheTrisbufferatthesametemperatureatwhichitwillbeused,asthepHvarieswithtemperature(about0.028pHunitsper1°C).

          RT
          Loadingbuffer10x

          		Prepareconcentratedstocksolutionofloadingbufferbyaddingthefollowingreagentsattheindicatedfinalconcentrations:20%Ficoll400,0.1MEDTA(pH8.0),1%SDS,0.25%bromophenolblue,0.25%xylenecyanol(optional).
          RT
          TBEbufferstocksolution

          		Dissolvein800mlofH2O108gTrisbase(89mM),55gboricacid(89mM),40ml0.5MEDTA,pH8.0(2mM);bringto1literwithH2O.Usediluted1:10.
          RT
          EthidiumbromidestocksolutionDissolve50mgofethidiumbromidein100mlofH2O.Usediluted1:1000.
          4°C
          Protectfromlight.
          AgarosegelDissolve1%agarosein1xTBEbuffer(inthepresenceof0.5mg/mlethidiumbromide)byheatinguntilmelted.
          Preparejustbeforeuse.
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