杨荣武分子生物学课件Week2modification.ppt

DNAandRNAisolation purification visualizationandquantitation GenomicDNApreparationoverviewPlasmidDNApreparationDNApurificationPhenolextractionEthanolprecipitationRNAwork WhatdoweneedDNAfor Detect enumerate clonegenesDetect enumeratespeciesDetect sequencespecificDNAregionsCreatenewDNA constructs recombinantDNA WhataboutRNA Whichgenesarebeingtranscribed When wherearegenesbeingtranscribed Whatistheleveloftranscription cellgrowth cellharvestandlysis DNApurification DNApurification overview DNAconcentration BacterialgenomicDNAprep cellextract Lysis DetergentsOrganicsolventProteases lysozyme Heat cellextract GenomicDNAprep removingproteinsandRNA AddtheenzymeRNasetodegradeRNAintheaqueouslayer Needtomixgently toavoidshearingbreakageofthegenomicDNA chloroform 2waystoconcentratethegenomicDNA 70 finalconc spooling Ethanolprecipitation GenomicDNAprepinplants howgetridofcarbohydrates CTAB Cationicdetergent MC6 61 6 62 lowionicconditions N CH3 Br CH3 CH3 C16H33 Plasmids vehiclesofrecombinantDNA Bacterialcell genomicDNA plasmids Non chromosomalDNAReplication independentofthechromosomeManycopiespercellEasytoisolateEasytomanipulate Plasmidpurification alkalinelysis AlkalineconditionsdenatureDNANeutralize genomicDNAcan trenature plasmidsCANbecausetheyneverfullyseparate DNApurification silicabinding Bindingoccursinpresenceofhighsaltconcentration andisdisruptedbyelutionwithwater DNApurification phenol chloroformextraction1 1phenol chloroformor25 24 1phenol chloroform isoamylalcoholPhenol denaturesproteins precipitatesformatinterfacebetweenaqueousandorganiclayerChloroform increasesdensityoforganiclayerIsoamylalcohol preventsfoaming Aqueousvolume atleast200microliters Add2volumesofphenol chloroform mixwellSpinincentrifuge moveaqueousphasetoanewtubeRepeatsteps2and3untilthereisnoprecipitateatphaseinterface extractaqueouslayerwith2volumesofchloroform Phenolextraction EthanoldepletesthehydrationshellsurroundingDNA AllowingcationstointeractwiththeDNAphosphatesReducingrepulsiveforcesbetweenDNAstrandsCausingaggregationandprecipitationofDNAAqueousvolume example 200microliters add22microliterssodiumacetate3MpH5 2 add1microliterofglycogen givesavisiblepellet add2volumes 446microliters 100 ethanol mixwell centrifugeathighspeed decantliquid washpellet 70 ethanol drypellet dissolveinappropriatevolume thendetermineDNAconcentration Ethanolprecipitation DNAconcentration cellgrowth cellharvestandlysis DNApurification DNApurification overview DNAconcentration IsolationofRNA Coursereading11 DNA mRNA protein LotsofinformationinmRNA Whenisgeneexpressed Whatistimingofgeneexpression Whatisthelevelofgeneexpression butwhatdoesanmRNAmeasurementreallysayaboutexpressionoftheprotein RNAinatypicaleukaryoticcell 10 5microgramsRNA80 85 isribosomalRNA15 20 issmallRNA tRNA smallnuclearRNAs About1 5 ismRNA variableinsize butusuallycontaining3 polyadenylation Theproblem s withRNA RNAischemicallyunstable spontaneouscleavageofphosphodiesterbackboneviaintramoleculartransesterificationRNAissusceptibletonearlyubiquitousRNA degradingenzymes RNases RNasesarereleaseduponcelllysisRNasesarepresentontheskinRNasesareverydifficulttoinactivate disulfidebridgesconferringstability norequirementfordivalentcationsforactivity CommonsourcesofRNaseandhowtoavoidthem Contaminatedsolutions buffersUSEGOODSTERILETECHNIQUETREATSOLUTIONSWITHDEPC whenpossible MAKESMALLBATCHESOFSOLUTIONSContaminatedequipmentUSE RNA ONLY PIPETS GLASSWARE GELRIGSBAKEGLASSWARE 300 C 4hoursUSE RNase free PIPETTIPSTREATEQUIPMENTWITHDEPC Top10sourcesofRNasecontamination AmbionScientificwebsite UnglovedhandsTipsandtubesWaterandbuffersLabsurfacesEndogenouscellularRNasesRNAsamplesPlasmidprepsRNAstorage slowactionofsmallamountsofRNAseChemicalnucleases Mg2 Ca2 at80 Cfor5 Enzymepreparations InhibitorsofRNaseDEPC diethylpyrocarbonatealkylatingagent modifyingproteinsandnucleicacidsfillglasswarewith0 1 DEPC letstandovernightatroomtempsolutionsmaybetreatedwithDEPC addDEPCto0 1 thenautoclave DEPCbreaksdowntoCO2andethanol InhibitorsofRnaseVanadylribonucleosidecomplexescompetitiveinhibitorsofRNAses butneedtoberemovedfromthefinalpreparationofRNAProteininhibitorsofRNasehorseshoe shaped leucinerichprotein foundincytoplasmofmostmammaliantissuesmustbereplenishedfollowingphenolextractionsteps MakingandusingmRNA 1 MakingandusingmRNA 2 PurifyingRNA thekeyisspeedBreakthecells solubilizecomponents inactivateRNAsesbytheadditionofguanidiniumthiocyanate verypowerfuldenaturant ExtractRNAusingphenol chloroform atlowpH PrecipitatetheRNAusingethanol LiClStoreRNA inDEPC treatedH20 80 C informamide deionized at 20 C SelectivecaptureofmRNA oligodT celluloseOligodTislinkedtocellulosematrixRNAiswashedthroughmatrixathighsaltconcentrationNon polyadenylatedRNAsarewashedthroughpolyARNAisremovedunderlow saltconditions notallofthenon polyadenylatedRNAgetsremoved OthermethodstocapturemRNAPoly U sepharosechromatographyPoly U coatedpaperfiltersStreptavidinbeads AbiotinylatedoligodTisaddedtoguanidinium treatedcells anditannealstothepolyAtailofmRNAsBiotin streptavidininteractionspermitisolationofthemRNA oligodTcomplexes HowgoodistheRNAprep TherRNAshouldform2sharpbandsinethidiumbromide stainedgels butmRNAwillnotbevisibleUseradiolabelledpolydTinapilotNorthernhybridization shouldgetasmearfrom0 6to5kbontheblotUseaknown standard geneprobe e g GAPDHinmammaliancells inNorthernhybridization thereshouldbeasharpbandwithnodegradationproducts VisualizingDNAandRNA non specificmethods QuantitationofDNAElectrophoresisVisualizingDNAingels VisualizingDNA Electrophoresis Allowsseparationofbiomolecules DNA RNA protein onbasisofsizeAseparationmatrix orgel agaroseorpolyacrylamide issaturatedwithanelectricallyconductivebufferSamplesareloaded anelectricfieldisapplied andnegativelychargedbiomoleculesinthesampletraveltowardthecathodeThelargerthemolecule theslowerthetravelthroughthegelmatrixDyesallowavisualestimateoftherateoftravelthroughthegelThechoiceofmatrixdependsmainlyonthesizeofDNAbeinganalyzed Agarosegels Agarose apolysaccharidepolymerofalternatingD andL galactosemonomers isolatedfromseaweedPoresizeisdefinedbytheagaroseconcentration higherconcentration slowerDNAmigrationoverall TheconformationoftheDNA supercoiled nickedcircles linear affectsthemobilityoftheDNAingelsRateofDNAmigrationisaffectedbyvoltage 5to8Volts cmisclosetooptimal Agarosecomesinamyriadoftypes variablemeltingtemperatures generatedbydifferentialhydroxyethylationoftheagarose Agarosegels StandardgelscanseparateDNAfragmentsfrom100bptoabout20 000bpPulsed fieldgelsseparateverylargeDNAfragments upto10 000 000bp or10Mb ThisapparatusallowsperiodicshiftsinthedirectionofDNAmigration 120 referstothereorientationangle differencebetweenorientationofelectricfieldsAandB timeofelectrophoresis progressmonitoredbymarkerdyes Loadsamplesinwells bromophenolBlue 500bp xyleneCyanol 4000bp Typicalagarosegel theDNAfragmentsarenotvisiblewithoutsomesortofstaining Polyacrylamidegels Acrylamidemonomers toxic polymerizedtoformgelmatrixThegelstructureisheldtogetherbythecross linker usuallyN N methylenebisacrylamide bis forshort Poresizedefinedbyconcentrationofgel totalpercentage andconcentrationofthecrosslinker bis relativetoacrylamidemonomerVeryhighresolution betterthanagarose Suitableforseparationofnucleicacidsfrom6to1000basepairsinlength Polyacrylamidegels Nativegels DNAstaysdouble stranded Denaturinggels runinthepresenceofhighconcentrationsofdenaturant usuallyurea andathightemperature DNAissinglestranded sequencinggels alsousefulinseparationofproteins whenproteinsaretreatedwithSDS whichdenaturesproteinsandgivesauniformlynegativesurfacecharge Recipeforapolyacrylamidegel Acrylamide anywherefrom4to20 dependingsizeofnucleicacidsorproteinsinthegel Bis acrylamide theratioofBistoregularacrylamideisimportant WaterBufferToinitiatepolymerization addAPS Ammoniumpersulfate generatesfreeradicalsneededforpolymerizationTEMED N N N N tetramethylethylenediamine acceleratesfreeradicalgenerationbyAPS MoreaboutgelsTherehastobeabuffer forcarryingcurrent TAE Tris acetate EDTA goodresolutionofDNA butbufferingcapacityisquicklydepletedTBE Tris borate EDTA Highbufferingcapacity resolutionisprettygoodUsegelloading buffers relativelysimple Densematerialtocarrysampletobottomofwells sucrose glycerol orficoll DyesfortrackingprogressofelectrophoresisBromophenolblue fastmigrationXylenecyanol slowmigrationOccasionallydenaturantispresent formamide fordenaturinggels e g sequencinggels ethidiumbromide ananti trypanosomaldrugforcattleStainworksbyintercalatinginstackedbasepairs elongatesDNAhelixFluorescenceincreasesuponDNAbindingStainedbandsvisualizedbyUVillumination 302or260nm Stainingnucleicacids Ethidiumbromide G Cbasepair Exampleofanagarose DNAgel Stainedwithethidiumbromide Directionofelectrophoresis Fragmentsofbacteriophage genomicDNA 48kb cutwiththerestrictionenzymeHindIIIThefragmentsareequimolar whyisthebandintensitydifferent Anotherethidiumbromide stainedagarosegel Themarkerlane M givessizestandardsforcomparisonwiththesamplelanes M samples OthermethodsforstainingDNA SYBRgold MolecularProbes Eugene OR morethan10 foldmoresensitivethanethidiumbromidefordetectingDNA butexpensive methyleneblue nottoxic butthestainingprotocolistimeconsuming andsensitivitysomewhatlowerthanethidiumbromidesilverstaining highdegreeofsensitivity buttheprotocolsaretimeconsuming andproteinsarealsostainedbysilver Southernblots DNA DNAhybridizationB Northernblots DNA RNAhybridization Microarray MethodsfordetectingspecificDNA RNA VisualizingDNA RNAandProtein detectingspecificsequences Techniquesallowonetodistinguishspecificsequencesorproteinsinalarge mixedpopulation e g incellextractsorgenomicDNApreparationsForDNAandRNA specificsequencedetectionisbasedonDNAandRNAcomplementarityandbase pairing DetectingspecificDNAsequences theSouthernblot AgaroseorPolyacrylamidegel nitrocelluloseornylonmembraneboundary DNAbindstoit Atypicalcapillaryblottingapparatus Electroblottingisalsocommonlyused Immobilizationofnucleicacids DNAisfixedtothenylonmembraneby Baking 80 CUVcrosslinking linksthyminesinDNAto chargedaminegroupsinmembrane DNAonlyProbetodetectsequenceofinterestbybase pairing hybridization ObtainprobeDNA syntheticoligonucleotideorclonedgene singlestranded LabelprobeforlaterdetectionRadioactivityNon radioactivelabel Southernblotting ImmobilizationoftargetDNAanddetection UseT4polynucleotidekinase catalyzesthetransferofthegammaphosphateof32PATPtothe5 endofDNAfragmenttobeusedasaprobe32Pisahighenergybetaparticleemitter andprovidesgoodsensitivityfordetectionofhybridizationbetweentheprobeDNAandthetarget blot DNADetectradiolabelwith autoradiography Xrayfilm phosphorimager phosphorcoatedplatesstoretheenergyoftheradioactiveparticle laserexcitationreleasesphotonsoflightthatarecollectedandrepresentedasapicture greaterdynamicrangethanfilm andfastertoo Radioactiveprobes 32Plabeling e g horseradishperoxidase oxidation Non radioactivelabels ordigoxygenin antibody conjugatedHRP canalsousebiotinylatedDNAprobe oxidation Non radioactivelabels blockingagents e g milk SDS preventnon specificinteractionsbetweenprobesandmembraneVolumeexclusionagents eg dextransulfate increaserateandlevelofhybridizationWashblotwithincreasingstringency Lowstringency highsalt lowtemperature probebindstosequenceswithmismatchesHighstringency lowsalt highertemp probebindsonlytofullycomplementarysequences Hybridizeprobestomembranes SouthernBlot oneexample RFLPs orPCRfragment SamebasictechniqueasSouthernblots butRNAisrunontheinitialgelandistransferredtothemembrane Usethismethodtomeasurelevelsofgenetranscriptioninvivo detectingchangesinthelevelsofRNAtranscriptunderdifferingconditions MicroarraysformeasuringmRNAabundancearebasedonthisprinciple butmanyprobesareimmobilizedinaregulararray reversetranscribed andfluorescentlylabelled RNA lightsup theprobesonthemicroarray Northernblots QuantitationofDNAbyUVabsorbance MeasureabsorbanceofUVlightbysample thearomaticbaseshaveacharacteristicabsorbancemaximumataround260nanometers 1 0A260 1cmlightpath DNAconcentrationof50microgramsperml doublestrandedDNA or38microgramsperml single strandedDNAorRNA theeffectiverangeforaccuratemeasurementisrathernarrow A260from0 05to2 0 DNAconcentrationsfrom2 5to100micrograms ml Samplemustbeverypureforaccuratemeasurements RNA EDTAandphenolallabsorbat260nm 0 5 0 Absorbance 1cmpathlength Wavelength nm 200 260 400 Atypical good scan multiplewavelengths ofaDNAsample A260 0 327 A260 A280 1 8isgood lowervaluesindicatesignificantproteincontamination Example sampleof250basepairfragmentofDNAhasanA260 327Whatisitsmolarconcentration Given 1 0A 50micrograms mlDNA DNAconc 327x50 16 35micrograms mlMWofanaveragebp 650DaltonsTherefore250bp FragmenthasaMWof1 6x105DaltonsSolveformolarity 1 02x10 7M or102nanomolar nM Importanttoknowhowtodothiscalculation HowdoesA260giveyouthequantityofDNA 1ml 16 35micrograms 1000ml 1L 106micrograms 1gram Whatisthemolarityofa16 35microgram mlsolutionofa250basepairDNAfragment 1 6x105grams 1mole 1 02x10 7molar0 102x10 6molar 0 1micromolar M 102x10 9molar 102nanomolar nM Fluorometry anothermethodforquantitationofDNAHoechst33258 afluorescentdye BindstoDNAintheminorgroove withoutintercalation FluorescenceincreasesfollowingbindingGoodforquantitationoflowconcentrationsofDNA 10 250ng ml rRNAandproteindonotinterfereButyouneedafluorometer AnothermethodforquantitationofDNA Ethidiumbromide fluorescentdye bindingComparesampleDNAfluorescencetostandardsofknownconcentration dilutionseries Insolution or usinggelelectrophoresis AcommerciallyavailablequantitativeDNAstandard