分子生物學學習資料：MB answers 05

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Chapter5MolecularToolsforStudyingGenesandGeneActivity

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DNAloadedinwells

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SDS,sodiumdodecylsulfate,isadetergentusedinSDSpolyacrylamidegelelectrophoresis.SDSdenaturestheproteinsandseparatesthesubunitsofcomplexproteins.Inaddition,itcoatsallthepolypeptideswithanegativechargesothattheymigrateaccordingtotheirmolecularmassesandnottheirnaturalcharge.

SDSandmoderntwo-dimensionalgelelectrophoresisbothseparateproteinsaccordingtotheirmolecularmasses.However,intwo-dimensionalgelelectrophoresis,theproteinsaresubjectedtoisoelectricfocusingpriortoseparationbySDS.Intheisoelectricfocusingprocess,theproteinsareelectrophoresedinapHgradient.Proteinswillstopmigratingwhentheyreachtheirisoelectricpoint.ThegelfromthetubeisthenplacedonthetopofanSDSgelandsubjectedtoseparationaccordingtomolecularmass.Twodimensionalgelelectrophoresisthereforeseparatesproteinsbasedonboththeirmassandisoelectricpoint.SDSseparatesproteinssolelybasedontheirmolecularmass.

Ionexchangechromatographyseparatesproteinsbasedontheirnetcharge.Inthisprocedureacomplexproteinmixtureisloadedontoachargedresin.Inthecaseofanionexchangechromatographythisisapositivelychargedresin.Solutionsofincreasingionicstrengtharepassedoverthecolumnandtheionsinthesesolutionscompetewiththeproteinsforbindingsitesontheresin.Proteinswithlesschargeareelutedatlowerionicstrengththanthosewithagreatercharge.

Gelfiltrationchromatographyseparatesproteinaccordingtotheirphysicaldimensions.Itinvolvespassingtheproteinsthroughaporousresininacolumn.Theresinhasholesofadefinedsize.Smallerproteinsemergemoreslowlythroughthecolumnthanlargeronesbecausetheirsmallerdimensionallowsthemtopassintotheholesintheresingivingthemalongerpathtotravel.Largerproteins,ontheotherhand,willnotbeabletoenterthebeadsandwillemergemorerapidly.Intermediatesizedproteinswillentersomebeadsandnotothersandwillemergeafteranintermediateperiodoftime.

Autoradiographyandphosphorimagingarebothtechniquesthatallowvisualizationofaradioactivesignalimmobilizedonamembrane.Bothdetecttheβ-particlesemittedfromtheisotopescommonlyusedinmolecularbiology.Inthecaseofautoradiography,theemittedparticlesexposetheemulsiononanx-rayfilm,leavingdarkbands.Autoradiographyislimitedinitsabilitytoquantifytheamountofisotopeexposingthefilm.Thereasonforthisisthattheresponseoffilmtoradiationisnon-linearandsaturationofthesignalisoftenaproblem.Forexample,asamplewithsay10,000dpm(disintegrationsperminute)mayexposetheemulsiononthefilmtoitsmaximumcapacityandthereforeanothersamplewith50,000dpmwillgiveabandofthesameintensityonthex-rayfilm.Phosphorimaginginvolvesuseofaphosphorimagingplatethatcontainsmoleculesthatareexcitedbyexposuretoβ-particles.Theseexcitedmoleculesaccumulateovertimeandstayinanexcitedstateuntiltheyarescannedwithalaser.Theenergyisthenreleasedandconvertedtodigitalinformationthatcanbeaccuratelyquantified.Thedigitalsignalfromaphosphorimagerisdirectlyproportionaltothenumberofdpmemanatingfromtheparticularregiononthesamplebeingexposed.

Onenon-radioactivemethodfordetectingaparticularnucleicacidfragmentinanelectrophoreticgeltakesadvantageofthestrongnon-covalentinteractionbetweenthemoleculeavidinandbiotin.Todetectaspecificfragmentinthegel,itisblottedtoamembrane(nitrocelluloseornylon)thusimmobilizingthenucleicacidfragmentonasolidsupport.Allofthesitesonthefilterthatareunboundbynucleicacidareblockedbytheadditionofnon-specificDNA.AprobecomplementarytothenucleicacidtargetissynthesizedinthepresenceofdUTPlabeledwiththevitaminbiotin.Theprobeisdenaturedandhybridizedtothenucleicacidsonthemembranewhereiswillbinditscomplementarysequence.Thenucleicacidprobehybridsaredetectedbyexposingthemembranetoareagentcontainingavidinlinkedtoanenzymesuchasalkalinephosphatase.Theprobeisvisualizedbytheactionoftheenzymeonaphosphorylatedsubstratewhichyieldsachemiluminescentcompoundthatisthendetectedbyanx-rayfilm.

ThediagramaboveillustratedtheprocessofSouthernblottingtodetectaDNAfragmentofinterestfromwithinacomplexmixtureoffragments.Inthisprocedure,specificDNAfragments,separatedonthegel,arevisualizedbyhybridizationoftheDNAwithalabeledprobe.TheNorthernblottingprocedureisusedtodetectaparticularRNAofinterestfromwithinacomplexmixtureofRNAmolecules.BothNorthernblottingandSouthernblottingrequireelectrophoresistoseparatethemixtureofnucleicacidsbysize.However,forSouthernblottingweneedtodigesttheDNAwithrestrictionenzymestogeneratesmallernucleicacidpiecesforelectrophoresis.TheRNAmoleculesloadedonthegel(eithertotalRNAorpoly(A)+RNA)alreadyexistassmallerfragmentofdiscretelengths.Bothtechniquesrequireblottingofthenucleicacidfragmentsonthegeltoafilterandhybridizationofthisnucleicacidwithalabeledprobe.

DNAfingerprintingcanbeusedtodistinguishamongdifferentindividualsbasedonthecharacteristicnumberofrepeatspresentatmultiplelociinthegenomecalledminisatellites.Theseminsatellitescontainrepeatedsequencesandthenumberofrepeatedsequencesataparticularlocusishighlyvariableinthepopulation.Togenerateafingerprintofanindividual,genomicDNAiscutwitharestrictionenzymespecificallychosenbecauseitdoesnothavearecognitionsitewithintherepeatedsequence.ASouthernblotisperformedusingtherepeatedsequenceasaprobe.Foranyoneindividual,multiplebandsrepresentingfragmentsofdifferentlengthwillbeseenontheblotrepresentingmultipleloci.Individualscanbetypedbasedontheiruniquenumberofrepeatsatthedifferentloci.DNAfingerprintsgeneratedinthisfashioncanhoweverbecomplextointerpret.Thisisduetothelargenumberofbandsontheblotwhichmaynotallresolvewell.ModernforensicmethodsthereforeuseDNAtypingwhichanalyzespolymorphismsasinglelocusatatime.PolymorphismsinthesesinglelocicanbedetectedusingRFLPanalysis(restrictionfragmentlengthpolymorphisms).RFLP’stakeadvantageofdifferentrestrictionpatternsthatmaybegeneratedduetobasepairsubstitutionintheDNAsequence.LikeDNAfingerprinting,RFLPanalysistypicallyusesSouthernblotting.Alternatively,differentfragmentlengthsindifferentindividualsatindividuallocimaybedetectedbyPCR.PCRallowstypingofverysmallamountsofDNAoflowquality.Fingerprinting,ontheotherhand,requiresmoreDNAofhigherqualityforSouthernblottingthandoesPCR-basedDNAtyping.Ofcourse,toreliablyuseDNAtypingdatainsuspectidentificationmultiplelocineedtobeanalyzedtoprovideahighprobabilitythatamatchbetweenasampleandanindividualisnotattributabletochancealone.

ANorthernblotcanbeusedtodetectaspecificRNApresentinatotalRNAsample.Fromthiswecantellifageneisbeingtranscribedinaparticularcelltypeorothersample.Inaddition,itcanprovidequantitativedataaboutthesteadystateRNAlevelpresentforaparticularRNAanditfurthergivesusthesizeoftheRNAtranscript.

FluorescenceinsituhybridizationallowsustousealabeledprobetodetectaparticularDNAsequenceonachromosome.AllofthechromosomesinacellarespreadandpartiallydenaturedtoallowtheDNAtohybridizetoasingle-strandedprobe.VisualizingtheprobecantellusthelocationorlocationsoftheDNAsequenceonthechromosome.Therefore,unlikeaSouthernblot,itgivesusinformationaboutthepositionofthegeneinthechromosome.Itisthereforeusefulwhenmapdataarerequired.

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ThenucleotidesequenceoftheDNAnucleotidechainsynthesizedfromtheprimeris:

5’GATGGCTAAATGTCTGACTTAATC3’

Therefore,thenucleotidesequenceoftheDNAnucleotidechainusedasthetemplatestrandisthereversecomplementofthesequenceabove:

5’GATTAAGTCAGACATTTAGCCATC3’

SangerDNAsequencingcanreadilybeautomatedusingdideoxynucleotideseachlabeledwithadifferentfluorescenttag.Thenestedsetoffragmentsgeneratedbythesequencingreactionsareseparatedbygelorcapillaryelectrophoresis.Asfragmentsprogressthroughthegel,alaseratthebottomofthegeldetectsthem.Thelasercandistinguishbetweenthefourdideoxynucleotidesanddeterminethesequenceofthenewlysynthesizedstrand.

Forthe1.5kbpEcoR1,XbaIDNAfragmentdepictedbelow,wecandistinguishorientationAfromorientationBusingtherestrictionmaptointerpretadoubledigest.AcloneinorientationAwillresultinfragmentsizesof3.3kband1.2kbafterdigestionwithXbaIandBamI.AcloneinorientationBwillgivefragmentsizesof4.2kband0.3kbpupondigestionwithBamH1andXbaI

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Site-directedmutagenesisisatechniquethatcanbeusedtogenerateaDNAsequencethathasbeenmodifiedatonespecificbase.Ingeneral,site-directedmutagenesisisaccomplishedbydesigningaprimerwiththeappropriatenucleotidechangethatwill,despitehavingamismatch,bindtothetargetwild-typesite.ReplicationofthetargetDNAwillleadtotheproductionofdaughterDNAmoleculeswiththealterednucleotidethathavebeenextendedfromtheprimer.OnespecificapproachisPCR-basedsite-directedmutagenesis.InthisprocesstheDNAfragmenttobealteredisclonedinaplasmidvectorandpropagatedinastrainofE.colithatmethylatestheA’sofGATCsequences.MutagenicprimersareannealedtothepurifiedplasmidcontainingthecloneandasmallnumberofroundsofPCRareperformedusingahighfidelity,heat-stablepolymerasesuchasPfupolymerase.Thisisimportanttoavoiderrorsincopyingtheclone.The

DNAinthePCRreactionisdigestedwiththerestrictionenzymeDpnIThisenzymewilldigestonlymethylatedor(lessefficiently)hemimethylatedDNAandwillspecificallydestroytheoriginaltemplateDNA.TheDNAproducedinvitrowithPfupolymeraseisnotmethylated,willnotbedigestedwithDpnIandcanbeusedtotransformE.coli.SequencinganumberofcloneswillidentifyoneswiththedesiredDNAnucleotidechange.

S1nucleaseanalysisallowsustolocatethe5’endorthe3’endofthetranscriptrelativetoaknownrestrictionsiteintheclone.ItreliesupontheuseofS1nuclease,anenzymespecificforsinglestrandedRNAinaDNA/RNAhybrid,todigestallunhybridizedRNAfromatranscripthybridizedwithacharacterizedknownsizegenomicfragment.TheintensityofthesignalproducedafterelectrophoresisandautoradiographyofthehybridsisproportionalthenumberoftranscriptspresentinthesampleandthereforeprovidesdataabouttherelativeamountsofRNApresent.PrimerextensiondoesnotusehybridizationoftheRNAtoaprobe.RatherisusesalabeledprimertoreversetranscribetheRNAtranscriptofinterestusingaprimercomplementarytoaregionwithinthegene.Thelengthofthefragmentproducedcantelluspreciselywhereupstreamoftheprimer-bindingsitethetranscriptended.Runningasetofdideoxysequencingreactions,usingthesameprimer,onthegelalongwiththeprimerextensionproductscanallowustolocatethe5’endofthetranscripttotheprecisenucleotide.LikeS1analysis,primerextensionassayscanallowustoquantifytheRNApresentinaparticularsample.PrimerextensionanalysisprovidesmoreaccurateinformationwhenmappingatranscriptsinceitavoidstheuseofnucleaseslikeS1.S1nucleasemayoccasionallyinappropriatelydigesttheendofadouble-strandedDNA/RNAhybridoritwilldigesttransientsingle-strandedstructuresintheRNA/DNAhybridresultingfrommeltingofATrichregions.ThistendencyofS1nucleasetowardsnon-specificdigestioncanresultsininaccuratesizingofthehybridsinthesetypesofanalysisresultingininaccurateinformationaboutthe3’and5’endsofthetranscript.OnlyS1mappingcanlocatethe3’-endofatranscript.Primerextensionwillnotworkbecausereversetranscriptiongoesonlyinthe3’5’direction.

Run-offtranscriptionisaninvitroassaythatmeasuresthesizeandabundanceoftranscriptsproducedfromagenomicDNAfragment.Agenomicfragmentthathasbeentruncatedataknownrestrictionsiteisusedasatemplatefortranscriptioninvitrowithalabelednucleotide.Thisallowsustodeterminewhetherthetranscriptsproducedareinitiatedatthecorrectlocationandtherelativeefficiencyoftranscriptioninitiatedfromaparticularpromoter.Thisassaycanthereforebeusedtoaskquestionsabouttheeffectofaparticularpromotermutationonthesizeoftranscriptsandtherelativeefficiencyoftranscription.However,becauseitisaninvitromethod,itcannotprovideanyinformationabouttheinvivolevelsoftranscription.

Labelingthe5’endofaDNAfragmentcanbeaccomplishedbyremovingtheterminalphosphatewithCalfIntestinalPhophatase(CIP)andthenusing[-32P]ATPandpolynucleotidekinasetointroducea5’phosphategroupthatisradioactivelylabeled.Labelingthe3’endofadoublestrandedDNAmoleculeisaccomplishedusingtheKlenowfragmentofDNApolymeraseanda[32P]dNTPtofillinarecessed3’OHendthatwasproducedbyrestrictionenzymedigestionofaDNAmolecule.

Anuclearrun-onassayisatechniquethatallowsustoacquirea“snapshot”ofthetranscriptionofaparticulargeneinvivo.Inotherwords,itallowsustoquantifyalltranscriptsthathavebeeninitiatedataparticulartime.Nucleiiareisolatedandthetranscriptionthathasbeeninitiatedisallowedtocontinueinthepresenceofalabelednucleotide.ReintiationoftranscriptionisinhibitedbythepresenceofheparintobindfreeRNApolymerase.TheextendedRNAproductscanbedetectedbyhybridizationtoacomplementaryprobeusingadotblot.

Adotblotallowsustoidentifyaspecificnucleicacid(RNAorDNA)sequencefromwithinacomplexmixturebyhybridizationofthetargettoaradiolabeledprobe.Itallowsustoquantifythehybrids.ThefundamentaldifferencebetweenadotblotandaSouthernblotisthatinadotblot,theDNAisnotseparatedbysizeusingelectrophoresisbeforehybridization.InsteadtheDNAissimplydottedonafilter.Itisthereforeusefulwhenthesizeofthetargetisunlikelytobeuniformorwhenitisnotnecessarytoknowthesizeofthetarget.

Reportergenescanbeusedtoreplaceanativegenewhenstudyingpromoterelementscontrollinggeneexpression.Insuchanexperimentthepromoterisaltered,andanumberofpromoterconstructsarelinkedtoareportergeneandreintroducedinthecell.Thelevelofexpressionofthereportergene(transcriptionandtranslation)thatisdirectedbythepromoterofinterestismeasured.Inthismannerwecanidentifyregionsofthepromoterthatareimportantindirectingexpressionofthegene.Therearetwoadvantagestothisapproach.First,thetranscriptiondirectedbytheendogenousgeneofinterestisnotgoingtointerferewithmeasurementsoftranscriptiondirectedbyintroducedgeneconstruct.Second,thereportergenessuchaslacZ,catorluciferasearechosenbecausetheyarereadilyassayable.

NitrocellulosefilterbindingassayscanbeusedtomeasurebindingbetweenaDNAandaprotein.Thesestudiestakeadvantageofthefactthatdouble-strandedDNAwillnotbindtoanitrocellulosefilterunlessitisassociatedwithaprotein.DNAislabeledandcombinedwithaproteinwithwhichitformsacomplex.Wecanmeasuretheamountofprotein-DNAcomplexthatisformedbymeasuringtheradioactivityboundtothefilter.Wecandeterminehowtightlytheproteinisboundbytestingtheabilityofnon-labeledtargetDNAtocompetewiththelabeledDNAforbindingoftheprotein.ThisisdonebyallowingthepurifiedproteintobindlabeledDNA,andthenaddingunlabeledcompetitorDNA.ThemixturesarefilteredthroughnitrocelluloseafterdifferentlengthsoftimeandscintillationcountingcanmeasuretheretainedlabeledDNA-proteincomplexes.

AgelmobilityshiftassaycandetectthespecificbindingofaproteintoasmallDNAfragment.Asthenameimplies,thebindingofaproteintoaDNAfragmentisvisualizedbyaretardationofthemobilityoftheDNAduringelectrophoresis.WhilethisprocedurecantelluswhetherornotaproteinisbindingaDNAfragment,itcannotgiveuspreciseinformationaboutwhichregionsornucleotideswithinthetargetDNAarebindingtheprotein.DNAsefootprintingontheotherhand,cantelluswherethetargetsitesforproteinbindingareontheDNA.TheproteinisboundtotheendlabeledDNAfragmentandtheproteinDNAcomplexesaresubjectedtoalimiteddigestwithDNaseIwhichwillresultinasetoffragments,eachcleavedonce(onaverage)withinthemolecule.ThesitesontheDNAthatareboundbyproteinwillhowevernotbecleavedandwhenthesefragmentsareelectrophoresedonageltherewillbeafootprintoragroupoffragmentsmissingontheautoradiograph.ThesemissingfragmentscorrespondtotheregionsontheDNAwheretheproteinisbound.BothtechniquesthereforecandetectbindingofaproteintoaDNAfragmentbutDNaseIfootprintinggiveusmorepreciseinformationonthelocationatwhichtheproteinisbound.

BothDMSandDNasefootprintingrelyonthefactthatifaproteinisboundtoaspecificregionofaDNAfragmentitwillpreventcleavageoftheDNAinthatregionbylimitingaccessofothermolecules.Forexample,itwilllimitaccessbythenuclease,DNaseI.DNaseIisalargemoleculeandmaynotbeabletodetectsmallgapscontainingunboundDNAthatmaybepresentinaDNA-proteincomplex.Inaddition,theassociationoftheproteinwiththeDNAmaydistortnearbyDNAregionsnotdirectlybindingtheprotein,andthesemaynotbedigestiblewithDNaseI.TogetamoredetailedpictureofwheretheproteinisboundtotheDNAwecanuseamethylatingagentsuchasdimethylsulfate(DMS),which,sinceitisasmallmolecule,willfitinthenooksandcranniesoftheDNA-proteincomplex,specificallymethylatingregionsnotassociatedwiththeprotein.InthesemethylationreactionsthereactiontimeandDMSislimitedsuchthatonaverage,onlyonemethylationeventwilloccurpermolecule.AfterremovingtheproteinwethentreattheDNAwithpiperidineandthiswillresultincleavageoftheDNA.TheprecisionwithwhichDMSaccessesfreeregionsofDNAascomparedtoDNaseIresultsinamoreprecisefootprintwhenusingDMSfootprinting.

Generatingaknockoutmousereliesupondetectingrarehomologousrecombinationeventsthatwillreplacearesidentgeneofinterestwithaninterruptedgeneconstructinmousestemcells.Markergenescanallowustodistinguishamongcellsthathaveundergonenorecombination,thoseinwhichtherehavebeennon-specificrecombinationevents,andthe“knockout”cellsinwhichtheresidentgenehasbeenreplacedbytheintroducedgene.Toaccomplishthisweengineerourclonedgeneofinterestsuchthatitscodingsequenceisinterruptedwithageneconferringresistancetotheantibioticneomycin.Inthesamevector,weengineerathymidinekinasegene.Stemcellsinwhichtherehavebeennorecombinationeventswillbesensitivetoneomycin,anditsderivative,G418,andcanbeselectedagainst.Cellsthathaveundergonenon-specificinsertionofvectorsequencesintorandomsitesinthegenomewilllikelyhaveincorporatedthethymidinekinasegenealongwiththeinterruptedgene.Wecanspecificallyselectagainstthesecellsalsobytakingadvantageofthefactthatcellscarryingathymidinekinasegene(tk+)cannotsurviveongangcyclovirwhereastheoriginalstemcells(whicharetk-)can.Specificrecombinationeventsinwhichtheresiden