《核酸研究方法》ppt课件

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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-chromosomal)p

2、hage/viral(dsorss)complementary(mRNA)GeneralFeatures:denaturingcelllysis(SDS,alkali,boiling,chaotropic)enzymetreatmentsproteaseRNase(DNase-free)DNase(RNase-free)CollectionofcellsUsesoftbrushestodislodgeepithelialcellsliningthemouth.Amplecellcollectionisverycriticalforsuccess.What’sinLysisBuffer?T

3、risbuffertomaintainpHofsolutionsothatDNAisstableSDStodissolvemembranesofcell,allowingDNAtobereleasedintosolutionSDSalsodenaturesproteins,makingthemmoresusceptibletoproteasecleavageLysisbufferOSOOO-SDSCH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH3-WhyaddProtease?Proteasedestroysnuclearproteinsthat

4、bindDNA,andcytoplasmicenzymesthatbreakdownanddestroyDNA(nucleases)ProteasetreatmentincreasestheyieldofDNAAddingsalt(5MNaCl)Na+bindstophosphategroupsofDNA,neutralizingtheelectricchargeofDNANaClallowsDNAmoleculesaggregateinsteadofrepellingeachother,makingiteasierforDNAtoprecipitateoutofsolutionwhena

5、lcoholisaddedAddingicecoldalcoholDNAcannotdissolveinalcoholTheadditionofcoldalcoholmakestheDNAclumptogetherandprecipitateoutofsolutionPrecipitatedDNAmoleculesappearaslongpiecesoffluffy,stringy,web-likestrandsMicroscopicoxygenbubbles“aggregate”or“fuse”together,simultaneouslywiththeDNAprecipitationT

6、helarger,visibleairbubblesactto“lift”theDNAoutofsolution,fromtheaqueousintotheorganicphaseDNAPrecipitationHighMWGenomicDNAIsolationTypicalProcedureCellLysis0.5%SDS+proteinaseK(55oseveralhours)PhenolExtractiongentlerockingseveralhoursEthanolPrecipitationRNAsefollowedbyproteinaseKRepeatphenolextrac-

7、tionandEtOHpptPhenolExtractionmixsamplewithequalvolumeofsat.phenolsolnretainaqueousphaseoptionalchloroform/isoamylalcoholextraction(s)aqueousphase(nucleicacids)phenolphase(proteins)HighMWGenomicDNAIsolationTypi