哈佛大学课件：DNA技术

TechniquestomanipulateDNAPhenol/chloroformextractionEthanolprecipitationConcentrationdeterminationGelElectrophoresisDNAhybridizationSouthernBlottingMeltingtemperaturesPCR免疫学信息网TechniquestomanipulateDNAPh1BRCA3EcoRIEcoRINIHExample:IstheBRCA3geneinvolvedinSuppressingbreastcancer?BRCA1BRCA2BRCA3免疫学信息网BRCA3EcoRIEcoRINIHExample:Is2PaperTowelsSouthernBlotting1tissueSDSProtKPhenolChloroETOHpptSpin免疫学信息网PaperTowelsSouthernBlotting13The“HyperchromicShift”2免疫学信息网The“HyperchromicShift”2免疫学信息4PaperTowelsSouthernBlotting1tissueSDSProtKPhenolChloroETOHpptSpin免疫学信息网PaperTowelsSouthernBlotting15AgaroseGelElectrophoresis_+3免疫学信息网AgaroseGelElectrophoresis_+36SampleWell25ng1kbladder0.8%Agarose124681012kbAgaroseGelElectrophoresisSybergoldDetectionLimit:~0.2-0.5ngDNAEtBrDetectionLimit:~5-10ngDNA(Notinhandout)免疫学信息网SampleWell124681012kbAgarose7Log(MW)islinearlyrelatedtomobility4100bpLow%hasnoResolvingpoweratlowMW1kb10kbHigh%hasnoresolvingpowerathighMW免疫学信息网Log(MW)islinearlyrelatedt8SeparationRangeVs.%Agarose5LowGelingAgarose4-6 0.02-0.4免疫学信息网SeparationRangeVs.%Agarose9TEMED(N,N,N,N-tetramethylenediamine)7免疫学信息网TEMED7免疫学信息网http://www.immune10PolyAcrylamideGels8免疫学信息网PolyAcrylamideGels8免疫学信息网htt11DNAtopologyaffectsmobilityofDNA9免疫学信息网DNAtopologyaffectsmobility12PFGEcanresolvelargeDNAfragments10免疫学信息网PFGEcanresolvelargeDNAfra13PaperTowelsSouthernBlotting1tissueSDSProtKPhenolChloroETOHpptSpin免疫学信息网PaperTowelsSouthernBlotting114HelixFormation:NucleationandPropagation11免疫学信息网HelixFormation:Nucleationa15A-TA-TG-CHelixMelting12免疫学信息网A-TA-TG-CHelixMelting12免疫学信息网16Whatparametersaffectthe

Tm?

(Tmisdefinedasthetempat

whichhalfthemolecules

aresingle-stranded)免疫学信息网Whatparametersaffectthe

Tm?17TmdependsonDNAlength13%dsDNASigmoidalCurve:HighlyCooperativeMelting!免疫学信息网TmdependsonDNAlength13%ds18Effectof%GCContentonTm14免疫学信息网Effectof%GCContentonTm1419Effectof[Salt]onTm15免疫学信息网Effectof[Salt]onTm15免疫学信息网20Predictingmeltingtemperatures(seelecturenotes)免疫学信息网Predictingmeltingtemperature21KineticsofHybridization:

DoIhavetimeforasodaora

week-endinNewYorkCity?免疫学信息网KineticsofHybridization:

Do22CotCurves:Hybridizationvs.Complexity16免疫学信息网CotCurves:Hybridizationvs.23ABi-PhasicCotCurve175kbRepeats(20%total)100kbfragmentRepeatDNA=5kbLongestnon-repeatDNA=100kb-20kb=80kbDifferenceincomplexity=80/5=16免疫学信息网ABi-PhasicCotCurve175kbRe24PaperTowelsSouthernBlotting1tissueSDSProtKPhenolChloroETOHpptSpin免疫学信息网PaperTowelsSouthernBlotting125ResultsofSouthernBlotControlTissueCancerTissueBRCA3EcoRIEcoRINIH免疫学信息网ResultsofSouthernBlotContro26BRCA3EcoRIEcoRINIH免疫学信息网BRCA3EcoRIEcoRINIH免疫学信息网http:2794C(30s)40-60C(30s)72C(30-60s/kb)MeltAnnealExtendThePolymeraseChainReaction(PCR)1820-30X免疫学信息网94C40-60C72CMeltAnnealEx2894C(30s)40-60C(30s)72C(30-60s/kb)MeltAnnealExtendCycle#2NIH20-30X免疫学信息网94C40-60C72CMeltAnnealEx29ResultsofPCR#1ControlTissueCancerTissueBRCA3EcoRIEcoRINIHPrimerSet#1免疫学信息网ResultsofPCR#1ControlTissu30ResultsofPCR#5ControlTissueCancerTissueBRCA3EcoRIEcoRINIHPrimerSet#5免疫学信息网ResultsofPCR#5ControlTissue31PCRmutagenesisEcoRIHindIIIIsolateproducts,mixAmplifydigest,subclonesequence20PvuII免疫学信息网PCRmutagenesisEcoRIHindIIIIso32免疫学信息网免疫学信息网http://www.immuneweb.co33LinearAmplificationGeometricAmplification19免疫学信息网LinearGeometric19免疫学信息网http:34hsp70EcoRIEcoRINIHExample:Isheatshockfactorbound