PCR-I-XXXX10-bin

Principle&PracticeofPCR罗彬Dept.Histolgy&EmbryologyWelcome!成绩评定实验课考勤笔试(开卷）课程安排理论课（6W-8W）：W2/3晚上实验课（9W-11W）：W1/2/4/5全天W3下午办公地点：104馆3楼科室电话：5358577Email：[e-mail],butpracticeiskeytoit.IntroductionaboutPCRPartI-WhatisPCR?-HowdidthePCRdevelop?-WhatdoweneedfordoingthePCR?-HowdidthePCRwork?-WhatisthePCRusedfor?Outline-WhatisPCR?-HowdidthePCRdevelop?-WhatdoweneedfordoingthePCR?-HowdidthePCRwork?-WhatisthePCRusedfor?OutlineWhatisPCR?PolymeraseChainReaction(聚合酶链反应)AmethodtoamplifyselectedsectionsofDNAinvitro(选择性体外扩增DNA片段的方法).Aprocesswhich“Amplifies”or“Copies”apieceofDNArepeatedlyuntilthereisanamountwhichisgreatenoughtoobservevisually.DNAreplicationvs.PCRPCRisalaboratoryversionofDNAreplicaitonandcommonlycalled“invitro”sinceitoccursinatesttubewhile“invivo”signifiesoccurringinalivingcell.-WhatisPCR?-HowdidthePCRdevelop?-WhatdoweneedfordoingthePCR?-HowdidthePCRwork?-WhatisthePCRusedfor?Outline*1930年正式提出核酸的概念*1953年Watson&Crick提出DNA双螺旋结构及半保留复制模型HarGobindKhorana*1971年Khorana最早提出核酸体外扩增的设想经过DNA变性，与合适的引物杂交，用DNA聚合酶延伸引物，并不断重复该过程便可克隆tRNA基因1968年诺贝尔生理学或医学奖“Idomybestthinkingwhiledriving”•Theideawasnottheproductofapainstakinglaboratorydiscipline,butwasconceivedwhilecruisingonhighway.•Forthisbrilliantidea,a$10,000bonusfromCetus.*1985年KaryMullis发明了PCR•receivedNobelPrizeforchemistryin1993.PCRhasbecomethemostwidelyusednucleicacidamplificationtechnologyandintegralpartofmolecularbiology.-fromthescientificmethodtoparapsychology,-frompoisonousspiderstotheHIVvirusandAIDS,-fromglobalwarmingtoastrology,-fromtheO.J.Simpsontrialtohowyoucanturnalightbulbonwithyourmind.DancingNakedintheMindFieldInthebookhewriteswithpassionandhumoraboutawiderangeofsubjects:-WhatisPCR?-HowdidthePCRdevelop?-WhatdoweneedfordoingthePCR?-HowdidthePCRwork?-WhatisthePCRusedfor?Outline5EssentialComponentsofPCRReactionsalts(ions)dNTPsPrimersDNAPolymeraseTemplateWhatdoweneedfordoingthePCR?BasicComponents:*Template(模板)：DNA,cDNA;*Primer(引物):oligonucleotides(寡核苷酸);*dNTP(三磷酸脱氧核苷酸)*DNAPolymerase(聚合酶)*Buffer(缓冲液)Areyouagoodcook?delicious•DeoxyribonucleicAcid(DNA)•ComplementaryDNA(cDNA):single-strandedDNAmadefromamessengerRNAtemplatewithreversetranscriptase(逆转录酶）.1.Template(模板)：Previouslyisolatedandpurified•ShortpreexistingoligonucleotidestowhichnewdeoxyribonucleotidescanbeaddedbyDNApolymerase•Twoprimershavetoflagthebeginningandendofthegenetobecopiedandarecomplementarytoit待扩增DNA片段两翼互补的寡核苷酸•Primersprovidespecificity5′3′3′5′Primer1Primer22.Primer(引物):oligonucleotides(寡核苷酸);TheSizeoftheDNAFragmentProducedinPCRisDependentonthePrimers•ThePCRreactionwillamplifytheDNAsectionbetweenthetwoprimers.•IftheDNAsequenceisknown,primerscanbedevelopedtoamplifyanypieceofanorganism’sDNA.ForwardprimerReverseprimerSizeoffragmentthatisamplified3.Deoxynucleotidetriphosphates（dNTP）:ProvideenergyandnucleosidesforthesynthesisofDNAAdenine（腺嘌呤）Thymidine（胸腺嘧啶）Cytosine（胞嘧啶）Guanine（鸟嘌呤）4.DNAPolymerase(聚合酶)Heat-stablepolymeraseisvitaltotheeaseoftheprocess…TaqPolymerase•AnenzymefromThermusaquaticusthatsurvivesinhotspringsinYellowstoneNationalParkandiscapableofgrowingin70-75℃；4.DNAPolymerase(聚合酶)•Originalreportwaspublishedin1976;•Roughly10yearslater,PCRwasdeveloped.•athermostableenzyme;•catalyzingthepolymerizationofnucleotidesintoduplexDNAinthe5´--3´direction;•5'–3'DNApolymerase;•lacking3'–5'exonucleaseactivity;havingerrorrate.•DNApolymeraseneedsMg++ascofactor;•EachDNApolymeraseworksbestunderoptimaltemperature,pHandsaltconcentration;•PCRbufferprovidesoptimalpHandsaltcondition.5.Buffer-WhatisPCR?-HowdidthePCRdevelop?-WhatdoweneedfordoingthePCR?-HowdidthePCRwork?-WhatisthePCRusedfor?OutlineHowdidthePCRwork?•3concepts•3steps•Onesentence3ConceptsofthePCR1.Denaturation:变性2.Renaturation：复性3.Semiconservativereplication：半保留复制thedoublestrandDNAopentosinglestrandedDNAtwosinglestrandedDNAformdoublestrandDNAreplication,onemaketwoSemi-conservativereplicationOldnew3stepsd-a-ecycle变性退火:time?/℃?延伸•openingdouble-strandDNAat94°C•Makingsingle-strandDNAaccessibletoprimerstemperature：94°~95°C•time：2~5mins(initialstep),30-60s(eachcycle）Denaturation/变性（Firststep）•UsealongertimeorhigherdenaturingtemperatureforGC-richtemplateDNA.•unnecessarilylongdenaturationtimeswilldecreasetheactivityofTaqDNAPolymerase.aprocessannealingprimerstothetemplate;•annealingtemperature（Ta）:55°~65°Cbasedonthemeltingtemperature（Tm）•annealingtime:30~60sAnnealing/退火(Secondstep)•PolymerasebindsandextendsacomplementaryDNAstrandfromeachprimer.•temperature:72oC(Taqworkingbest)•time:2min(eachcycle)/6min(finalextension)Extension/延伸(Thirdstep)ExtensionInitialdenaturingDenaturingAnnealing30-35cyclesFinalExtensionPCRThermocyclerAPCRmachinecontrolstemperaturePCRreactivecurveOnesentenceOnemakestwo,twomakefour,fourmakeanything-WhatisPCR?-HowdidthePCRdevelop?-WhatdoweneedfordoingthePCR?-HowdidthePCRwork?-WhatisthePCRusedfor?OutlineWhatisthePCRusedfor?•analysisofentiregenomes(基因组);•geneexpression(RT-PCR);•analysismutagenesisinvitro(体外);•todetectandquantifyminuteamountsofapathogen(病原体)…AdvantagesofPCRUsefulnon-invasiv