核酸的研究方法

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1、第十五章核酸的研究方法1、核酸的分离、提纯和定量测定DNA的分离RNA的分离核酸的定量IsolationofNucleicAcidsGoals:removalofproteinsDNAvsRNAisolationofaspecifictypeofnucleicacidTypesofMethods:differentialsolubility‘adsorption’methodsdensitygradientcentrifugationTypesofDNA:genomic(chromosomal)organellar(satellite)plasmid(extra-

2、chromosomal)phage/viral(dsorss)complementary(mRNA)GeneralFeatures:denaturingcelllysis(SDS,alkali,boiling,chaotropic)enzymetreatmentsproteaseRNase(DNase-free)DNase(RNase-free)CollectionofcellsUsesoftbrushestodislodgeepithelialcellsliningthemouth.Amplecellcollectionisverycriticalforsuc

3、cess.What’sinLysisBuffer?TrisbuffertomaintainpHofsolutionsothatDNAisstableSDStodissolvemembranesofcell,allowingDNAtobereleasedintosolutionSDSalsodenaturesproteins,makingthemmoresusceptibletoproteasecleavageLysisbufferOSOOO-SDSCH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH3-WhyaddProte

4、ase?ProteasedestroysnuclearproteinsthatbindDNA,andcytoplasmicenzymesthatbreakdownanddestroyDNA(nucleases)ProteasetreatmentincreasestheyieldofDNAAddingsalt(5MNaCl)Na+bindstophosphategroupsofDNA,neutralizingtheelectricchargeofDNANaClallowsDNAmoleculesaggregateinsteadofrepellingeachother

5、,makingiteasierforDNAtoprecipitateoutofsolutionwhenalcoholisaddedAddingicecoldalcoholDNAcannotdissolveinalcoholTheadditionofcoldalcoholmakestheDNAclumptogetherandprecipitateoutofsolutionPrecipitatedDNAmoleculesappearaslongpiecesoffluffy,stringy,web-likestrandsMicroscopicoxygenbubbles“

6、aggregate”or“fuse”together,simultaneouslywiththeDNAprecipitationThelarger,visibleairbubblesactto“lift”theDNAoutofsolution,fromtheaqueousintotheorganicphaseDNAPrecipitationHighMWGenomicDNAIsolationTypicalProcedureCellLysis0.5%SDS+proteinaseK(55oseveralhours)PhenolExtractiongentlerockin

7、gseveralhoursEthanolPrecipitationRNAsefollowedbyproteinaseKRepeatphenolextrac-tionandEtOHpptPhenolExtractionmixsamplewithequalvolumeofsat.phenolsolnretainaqueousphaseoptionalchloroform/isoamylalcoholextraction(s)aqueousphase(nucleicacids)phenolphase(proteins)HighMWGenomicDNAIsolatio

8、nTypi