基因工程第6章大分子的分離與分析基因工程第6章大分子的分離與分析1E.Coliplasmid

DNAIsolation,detectionandpurificationofDNAE.ColiplasmidDNAIsolation,2最新基因工程第6章大分子的分離與分析課件3最新基因工程第6章大分子的分離與分析課件4最新基因工程第6章大分子的分離與分析課件5最新基因工程第6章大分子的分離與分析課件6最新基因工程第6章大分子的分離與分析課件7最新基因工程第6章大分子的分離與分析課件8Polyacrylamidegelelectropholesis（PAGE）Advantages:highresolution(1bp)largesampleloading(10g)highpurityofrecoveredsampleNon-denature:

mobility(sequenceandcomposition)Denature(尿素/甲醛):isolateprobeanalyzetheproductsofenzymeS1DNAsequencingPolyacrylamidegelelectrophol9PurificationofDNAfragment

(fromagarose）Lowmeltingpointagarosegel+5voltris65℃5minphenolextractethanolPermeationbag(electricalelution)for5kbGlassMilk(bead)3MNaI，beadattachmenteluteKitQiagencorp.PurificationofDNAfragment10Atypicalmammalcell:10-5gRNA，but80-85%isrRNA(28S,18S,5.8Sandor5S(23S,16S,5S),and15～20%consistofsmallRNA（e.g.tRNA，hsRNAetc.）.1～5%oftotalRNAismRNA:3‘endpoly(A)tailadheretooligo(dT)-celluloseIsolation,purificationanddetectionofRNAAtypicalmammalcell:Isolatio11ControlofRNaseactivityExperimentaltoolsandsolutionglass,plastics,electrophoresisapparatus180℃8hrorlonger，chloroformwashing/NaOH0.1%DEPC(焦碳酸二乙酯)incubation(37℃,overnight)(stronginhibitorofRNase,butnotabsolutely)DEPC-treatedwaterwashingandautoclavingControlofRNaseactivity12Electrophoresisapparatus:

Runningwaterwashing,ethanoldrying3%H2O2×10min，0.1%DEPCH2OwashingH2O：0.1%DEPC（someagentscan’tbetreatedbyDEPC,e.g.Tris）otherssuchasgloves,cleanroomElectrophoresisapparatus:13ExtractionofRNAreferenceKitGibco/TR1ZOLReagent100ml99.8USD(酚+異硫氰酸胍)Qiagen/RNAeasyPurificationofRNARemovingproteinRemovingDNADNaseI（RNasefree）KitGradientcentrifugationExtractionofRNAPurification14PurificationofmRNAAffinitychromagraphyPuritydetectionOD260:1=40g/mlRNA

OD280,OD230ElectrophoresisAgrosegel氫氧化甲基汞甲醛乙二醛-二甲基亞礬（DMSO）PAGEPurificationofmRNAElectropho15ProteinsizesWesternblotN-terminalaminoacidsequencingSDSofproteinProteinsizesSDSofprote16Molecularhybridization(分子雜交)Molecularhybridization17TypesSouthern,Northern,Western,Dot,colony(plaque)Denature(變性)hot,extremepH,orgonicagents,ureaAnnealing(復性)complementaryssannealingtoformdsHybridization(雜交)homologousssannealingtoformdsTypes18SouthernblotDNAfragmentblotREdigestionelectrophoresis/photoneutral／alkaline（0.4N,NaOH）denature/neutralizeSouthernblot19blottingNitrocellulosemembraneNC(80-100μg/cm)nylonmembrane(chargedornot)(350-500μg/cm)6×SSC(氯化鈉和檸檬酸鈉)transferbuffer毛細管電轉移真空(真空轉移裝置)blotting20濾紙凝膠濾膜吸水紙玻璃板重物支持物500g<1kb<1hr,>15kb>18hr濾紙凝膠濾膜吸水紙玻璃板重物支持物500g<1kb21Crosslinking

80℃2hr,UV,microwaveCrosslinking22Hybridizationprehybridization(預雜交)6×SSCDenhardt(50x)：(withFicoll,聚乙烯比咯烷酮)denaturedfishspermDNA0.5%SDSHybridization23Hybridizationprobetreatment42℃50%甲酰胺6-8hrsprobe：DNA,Oligo,randomDNAMembranewashing(洗去未雜交的探針)2×SSC/0.5%SDS15minRT0.1×SSC/0.5%SDS1hrHybridizationMembranewashing(24最新基因工程第6章大分子的分離與分析課件25Detection

X-filmorBiochemicalmethods

Detection26最新基因工程第6章大分子的分離與分析課件27最新基因工程第6章大分子的分離與分析課件28最新基因工程第6章大分子的分離與分析課件29ProbelabelingEvenlylabeled(均一標記)nicktranslation(切口平移):

E.coliDNApolyI(DNaseI:14-16℃togeneratenick)randomprimer:(已取代前者)6Ntor6～12ntklenowssprobessDNAtemplate,universalprimer(notlikecDNA,RNaseH)RNAprobe，RNApolymeraseProbelabeling30EndlabelingKlenow3’end,補平和置換T4polymerase3’endstrong3’-5’exonuclease,esp.for3’protrudingendKinase5’end正反應32P→5’-OHNNNN→5’pNNNN交換反應overATP使5’-P→ADPthen32P→5’Endlabeling31Oligonucleotide

Kinase,

TerminaltransferaseLabelsRadio:32P，33P，35SNon-radio:DIGoxigenin,BiotintolabeldUTPOligonucleotideLabels32Example:

DIGlabel/detectionLabellingprobe6Ntoligoprimer(Klenow)Detectionwashingblockingimmunologicalreaction(labeledAnti-DiG-Ab-Ap(堿性磷酸酶)washingcolordeveloping/chemiluminescence(化學發光)NBT-BCIP形成棕黃色沉淀Example:DIGlabel/detection33BCIP：5-溴-4-氯-3’吲哚磷酸NBT:氯化硝基四氮唑藍（染料色素）CIP的底物,也稱氮藍四唑優點：無污染，方便，探針可重復使用，可控制顯色反應，保存雜交膜，易辯別真假陽性缺點：靈敏度不夠，不能多次雜交，0.1pg可檢測單拷貝，如g人胎盤DNA中的單拷貝BCIP：5-溴-4-氯-3’吲哚磷酸優點：無污染，方便，探34最新基因工程第6章大分子的分離與分析課件35最新基因工程第6章大分子的分離與分析課件36Insituhybridization

(原位雜交)

(colonyorplaque)Colonygrowthpositiveclonestwoplates(有/無膜)Replicatingmostcolonyorplaqueisrecombinants(whitecolor)DNAreleaseandcrosslinking

manymethods①10%SDS；②0.5NNaOH/1.5NNaCl③0.5MTris1.5NNaCl④SSC⑤干燥⑥固定80℃2hrInsituhybridization(原位雜交)D37Dothybridization(斑點雜交)DirectlyloadingDNAsampleonmembraneDothybridization(斑點雜交)38NorthernblotSimilartoSouthernblot,detectingtotalRNAormRNA.Electrophoresisbuffer甲醛buf(5×)0.1mMMOPS(pH2.0)(3-(N-瑪琳代)丙磺酸)40mMNaAc5mMEDTA(pH8.0)DEPCH2OGelmaking1×buf+2.2M甲醇（MW30.03,37%水溶液，12.3M（pH>4.0）Northernblot39LoadingRNAtreatment(upto30g）4.5l+5×buf2.0l甲醛3.5l甲酰胺10l65℃15minloadingbuffer50%甘油，1mMEDTA(pH8.0)0.25%BPB0.255二甲苯TFFRunninggelTransferSimilartoSouthernblotLoadingTransfer40WesternblotSimilartotheblotabovementionedKeydifference:antibodyasprobeMonoclonalantibodySpecificPolyclonalMixedantibodiesWesternblotSimilartotheblo41Steps:ProteinpreparationRunningSDSTransferring(nitrocellulosemembrane)StainingSteps:42Blockingfat-freemilk5%FirstantibodybindtargetproteinSecondaryimmunologicalreactionsecondantibody(抗免疫球蛋白抗體)proteinA

Labels：125I堿性磷酸酶辣根過氧化物酶DetectionBlockingfat-freemilk5%43RNAterminusanalysisEnzymeS1mapping(參見分子克隆指南,第558頁)Labeledoligo,PAGEgeltodetermineMW5‘3‘ATGUAGS1酶S1酶DetectingSplicingsiteofmRNAprecursor(內含子位置)RNAterminusanalysisLabeledo44Drawback

inmappingthe5’endsoftranscripts:Tendingto“nibble”abitontheendsoftheRNA-DNAhybrid,orevenwithinthehybridwhereA-T-richregionscanmelttransiently.Sometimes,tendingtonotdigestthesingle-strandedregionscompletely.

Drawbackinmappingthe5’end45Primerextension(引物延伸)Tolocatethe5′endofatranscriptbyhybridizinganoligonucleotideprimertotheRNAofinterest,extendingtheprimerwithreversetranscriptasetothe5′endofthetranscript,andelectrophoresisthereversetranscripttodeterminethesize.Theintensityofthesignalobtainedisameasureoftheconcentrationofthetranscript.Primerextension(引物延伸)46最新基因工程第6章大分子的分離與分析課件47基因工程第6章大分子的分離與分析基因工程第6章大分子的分離與分析48E.Coliplasmid

DNAIsolation,detectionandpurificationofDNAE.ColiplasmidDNAIsolation,49最新基因工程第6章大分子的分離與分析課件50最新基因工程第6章大分子的分離與分析課件51最新基因工程第6章大分子的分離與分析課件52最新基因工程第6章大分子的分離與分析課件53最新基因工程第6章大分子的分離與分析課件54最新基因工程第6章大分子的分離與分析課件55Polyacrylamidegelelectropholesis（PAGE）Advantages:highresolution(1bp)largesampleloading(10g)highpurityofrecoveredsampleNon-denature:

mobility(sequenceandcomposition)Denature(尿素/甲醛):isolateprobeanalyzetheproductsofenzymeS1DNAsequencingPolyacrylamidegelelectrophol56PurificationofDNAfragment

(fromagarose）Lowmeltingpointagarosegel+5voltris65℃5minphenolextractethanolPermeationbag(electricalelution)for5kbGlassMilk(bead)3MNaI，beadattachmenteluteKitQiagencorp.PurificationofDNAfragment57Atypicalmammalcell:10-5gRNA，but80-85%isrRNA(28S,18S,5.8Sandor5S(23S,16S,5S),and15～20%consistofsmallRNA（e.g.tRNA，hsRNAetc.）.1～5%oftotalRNAismRNA:3‘endpoly(A)tailadheretooligo(dT)-celluloseIsolation,purificationanddetectionofRNAAtypicalmammalcell:Isolatio58ControlofRNaseactivityExperimentaltoolsandsolutionglass,plastics,electrophoresisapparatus180℃8hrorlonger，chloroformwashing/NaOH0.1%DEPC(焦碳酸二乙酯)incubation(37℃,overnight)(stronginhibitorofRNase,butnotabsolutely)DEPC-treatedwaterwashingandautoclavingControlofRNaseactivity59Electrophoresisapparatus:

Runningwaterwashing,ethanoldrying3%H2O2×10min，0.1%DEPCH2OwashingH2O：0.1%DEPC（someagentscan’tbetreatedbyDEPC,e.g.Tris）otherssuchasgloves,cleanroomElectrophoresisapparatus:60ExtractionofRNAreferenceKitGibco/TR1ZOLReagent100ml99.8USD(酚+異硫氰酸胍)Qiagen/RNAeasyPurificationofRNARemovingproteinRemovingDNADNaseI（RNasefree）KitGradientcentrifugationExtractionofRNAPurification61PurificationofmRNAAffinitychromagraphyPuritydetectionOD260:1=40g/mlRNA

OD280,OD230ElectrophoresisAgrosegel氫氧化甲基汞甲醛乙二醛-二甲基亞礬（DMSO）PAGEPurificationofmRNAElectropho62ProteinsizesWesternblotN-terminalaminoacidsequencingSDSofproteinProteinsizesSDSofprote63Molecularhybridization(分子雜交)Molecularhybridization64TypesSouthern,Northern,Western,Dot,colony(plaque)Denature(變性)hot,extremepH,orgonicagents,ureaAnnealing(復性)complementaryssannealingtoformdsHybridization(雜交)homologousssannealingtoformdsTypes65SouthernblotDNAfragmentblotREdigestionelectrophoresis/photoneutral／alkaline（0.4N,NaOH）denature/neutralizeSouthernblot66blottingNitrocellulosemembraneNC(80-100μg/cm)nylonmembrane(chargedornot)(350-500μg/cm)6×SSC(氯化鈉和檸檬酸鈉)transferbuffer毛細管電轉移真空(真空轉移裝置)blotting67濾紙凝膠濾膜吸水紙玻璃板重物支持物500g<1kb<1hr,>15kb>18hr濾紙凝膠濾膜吸水紙玻璃板重物支持物500g<1kb68Crosslinking

80℃2hr,UV,microwaveCrosslinking69Hybridizationprehybridization(預雜交)6×SSCDenhardt(50x)：(withFicoll,聚乙烯比咯烷酮)denaturedfishspermDNA0.5%SDSHybridization70Hybridizationprobetreatment42℃50%甲酰胺6-8hrsprobe：DNA,Oligo,randomDNAMembranewashing(洗去未雜交的探針)2×SSC/0.5%SDS15minRT0.1×SSC/0.5%SDS1hrHybridizationMembranewashing(71最新基因工程第6章大分子的分離與分析課件72Detection

X-filmorBiochemicalmethods

Detection73最新基因工程第6章大分子的分離與分析課件74最新基因工程第6章大分子的分離與分析課件75最新基因工程第6章大分子的分離與分析課件76ProbelabelingEvenlylabeled(均一標記)nicktranslation(切口平移):

E.coliDNApolyI(DNaseI:14-16℃togeneratenick)randomprimer:(已取代前者)6Ntor6～12ntklenowssprobessDNAtemplate,universalprimer(notlikecDNA,RNaseH)RNAprobe，RNApolymeraseProbelabeling77EndlabelingKlenow3’end,補平和置換T4polymerase3’endstrong3’-5’exonuclease,esp.for3’protrudingendKinase5’end正反應32P→5’-OHNNNN→5’pNNNN交換反應overATP使5’-P→ADPthen32P→5’Endlabeling78Oligonucleotide

Kinase,

TerminaltransferaseLabelsRadio:32P，33P，35SNon-radio:DIGoxigenin,BiotintolabeldUTPOligonucleotideLabels79Example:

DIGlabel/detectionLabellingprobe6Ntoligoprimer(Klenow)Detectionwashingblockingimmunologicalreaction(labeledAnti-DiG-Ab-Ap(堿性磷酸酶)washingcolordeveloping/chemiluminescence(化學發光)NBT-BCIP形成棕黃色沉淀Example:DIGlabel/detection80BCIP：5-溴-4-氯-3’吲哚磷酸NBT:氯化硝基四氮唑藍（染料色素）CIP的底物,也稱氮藍四唑優點：無污染，方便，探針可重復使用，可控制顯色反應，保存雜交膜，易辯別真假陽性缺點：靈敏度不夠，不能多次雜交，0.1pg可檢測單拷貝，如g人胎盤DNA中的單拷貝BCIP：5-溴-4-氯-3’吲哚磷酸優點：無污染，方便，探81最新基因工程第6章大分子的分離與分析課件82最新基因工程第6章大分子的分離與分析課件83Insituhybridization

(原位雜交)

(colonyorplaque)Colonygrowthpositiveclonestwoplates(有/無膜)Replicatingmostcolonyorplaqueisrecombinants(whitecolor)DNAreleaseandcrosslinking

manymethods①10%SDS；②0.5NNaOH/1.5NNaCl③0.5MTris1.5NNaCl④SSC⑤干燥⑥固定80℃2hrInsituhybridization(原位雜交)D84Dothybridization(斑點雜交)DirectlyloadingDNAsampleonmembraneDothybridization(斑點雜交)85NorthernblotSimilartoSouthernblot,detectingtotalRNAormRNA.Electrophoresisbuffer甲醛buf(5×)0.1mMMOPS(pH2.0)(3-(N-瑪琳代)丙磺酸)40mMNaAc5mMEDTA(pH8.0)DEPCH2OGelmaking1×buf+2.2M甲醇（MW30.03,37%水溶液，12.3M（pH>4.0）Northernblot86LoadingRNAtreatment(upto30g）4.5l+5×buf2.0l甲醛3.5l甲酰胺10l65℃15minloadingbuffer50%甘油，1mMEDTA(pH8.0)0.25%BPB0.255二甲苯TFFRunninggelTransferSimilartoSouther