PCR聚合酶链反应英语介绍.doc

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1、VirologyTheBasicsTaxonomyLinksHaematologyDNADownUnderPCRDownUnderHOMEHeadlinesDNADownUnderNewsectiononRNAinterferenceasatooltoblockvirusreplication.Thepolymerasechainreaction(PCR)isatechniqueforcopyingapieceofDNAabillion-fold.Asthenamesuggests,theprocesscreatesachainofmanypieces,inthis

2、casethepiecesarenucleotides,andthechainisastrandofDNA.PCRisanenzyme-mediatedreaction,andaswithanyenzyme,thereactionmustoccurattheenzyme'sidealoperatingtemperature.TheenzymesthatareusedforthePCRareDNA-dependentDNApolymerases(DDDP)derivedfromthermophilic(heat-loving)bacteria.Assuch,theen

3、zymesfunctionathighertempertauresthantheenzymeswecommonlyuseinthelaboratoryorhaveworkinginourbodies.TheseDNApolymerasesoperateat60-75°C,andcanevensurviveattemperaturesabove90°C.ThisisimportantbecauseapartofthePCRrequiresthatthereactionreaches~95°Casweshallsee.ApartfromtheDNApolymerase,

4、PCRneedsaDNAtemplatetocopy,andapairofshortDNAsequencescalledoligonucleotidesor"primers"togettheDNApolymerasestarted.Broadlyspeaking,therearethreestepsidentifedbyincubatingatdifferenttempratures.thethreestepsmakeupaPCR"cycle".1Double-strandedDNAseparationordenaturation(DinFigure1)2Prime

5、rannealingtotemplateDNA(AinFigure1)3Primerextension(EinFigure1)Figure1.APCRcycle.Thethreetemperatureswhichmakeupasinglecycle.TheDNAdenaturatiuonsection(D),oligonucleotideannealingsection(A)andtheprimerextension(E)sectionaremarked.ThetemperaturerangeoverwhichdsDNAduplexescandenature(TD)

6、or'melt',andtherangeoverwhichtheoligonucleotideprimercanhybridise(TM)arealsomarked.DenaturationAttemperaturesabove90°C,double-strandedDNAdenaturesor"melts".Thatmeanstheweakhydrogenbondsthatusuallyholdthetwocomplementarystrandstogetheratnormaltemperaturesaredisruptedresultingintwosingle

7、strandedDNAstrands(shownbelowinanidealisedform).PrimerAnnealingAttheannealingtemperature(TA),primersthatcollidewiththeircomplementarysequencecanhybrdiseor"bind"toit.Thechanceofsuchanencounterhappeningisincreasedbecauseweuseavastexcessofeachprimerinthereactionmixturecomparedtothenumbe