RealTime PCRWord文档格式.docx
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TheadventofPolymeraseChainReaction(PCR)byKaryB.Mullisinthemid-1980srevolutionizedmolecularbiologyasweknowit.PCRisafairlystandardprocedurenow,anditsuseisextremelywide-ranging.Atitsmostbasicapplication,PCRcanamplifyasmallamountoftemplateDNA(orRNA)intolargequantitiesinafewhours.ThisisperformedbymixingtheDNAwithprimersoneithersideoftheDNA(forwardandreverse),Taqpolymerase(ofthespeciesThermusaquaticus,athermophilewhosepolymeraseisabletowithstandextremelyhightemperatures),freenucleotides(dNTPsforDNA,NTPsforRNA),andbuffer.ThismovieshowsPCRinaction.ThetemperatureisthenalternatedbetweenhotandcoldtodenatureandreannealtheDNA,withthepolymeraseaddingnewcomplementarystrandseachtime.InadditiontothebasicuseofPCR,speciallydesignedprimerscanbemadetoligatetwodifferentpiecesofDNAtogetheroraddarestrictionsite,inadditiontomanyothercreativeuses.Clearly,PCRisaprocedurethatisanintegraladditiontothemolecularbiologist’stoolbox,andthemethodhasbeencontinuallyimproveduponovertheyears.(Purves,etal.2001)
Fairlyrecently,anewmethodofPCRquantificationhasbeeninvented.Thisiscalled“real-timePCR”becauseitallowsthescientisttoactuallyviewtheincreaseintheamountofDNAasitisamplified.Severaldifferenttypesofreal-timePCRarebeingmarketedtothescientificcommunityatthistime,eachwiththeiradvantages.Thiswebsitewillexploreoneofthesetypes,TaqMan®
real-timePCR,aswellasgiveanoverviewoftheothertwotypesofreal-timePCR,molecularbeaconandSYBR®
Green.(2003)
HowTaqMan®
works:
TaqMan®
utilizesasystemthatisfairlyeasytograspconceptually.First,wemusttakealookattheTaqMan®
probe.
Figure1.TheTaqmanprobe.Theredcirclerepresentsthequenchingdyethatdisruptstheobservablesignalfromthereporterdye(greencircle)whenitiswithinashortdistance.ImagecreatedbyDanPierce.
Theprobeconsistsoftwotypesoffluorophores,whicharethefluorescentpartsofreporterproteins(GreenFluorescentProtein(GFP)hasanoften-usedfluorophore).WhiletheprobeisattachedorunattachedtothetemplateDNAandbeforethepolymeraseacts,thequencher(Q)fluorophore(usuallyalong-wavelengthcoloreddye,suchasred)reducesthefluorescencefromthereporter(R)fluorophore(usuallyashort-wavelengthcoloreddye,suchasgreen).ItdoesthisbytheuseofFluorescence(orFö
rster)ResonanceEnergyTransfer(FRET),whichistheinhibitionofonedyecausedbyanotherwithoutemissionofaproton.Thereporterdyeisfoundonthe5’endoftheprobeandthequencheratthe3’end.(2003)
Figure2.TheTaqMan®
probebindstothetargetDNA,andtheprimerbindsaswell.Becausetheprimerisbound,Taqpolymerasecannowcreateacomplementarystrand.ImagecreatedbyDanPierce.
OncetheTaqMan®
probehasboundtoitsspecificpieceofthetemplateDNAafterdenaturation(hightemperature)andthereactioncools,theprimersannealtotheDNA.TaqpolymerasethenaddsnucleotidesandremovestheTaqman®
probefromthetemplateDNA.Thisseparatesthequencherfromthereporter,andallowsthereportertogiveoffitsemititsenergy.Thisisthenquantifiedusingacomputer.Themoretimesthedenaturingandannealingtakesplace,themoreopportunitiestherearefortheTaqman®
probetobindand,inturn,themoreemittedlightisdetected.(2003)
Figure3.Thereporterdyeisreleasedfromtheextendingdouble-strandedDNAcreatedbytheTaqpolymerase.Awayfromthequenchingdye,thelightemittedfromthereporterdyeinanexcitedstatecannowbeobserved.ImagecreatedbyDanPierce.
Quantification:
Thespecificsinquantificationofthelightemittedduringreal-timePCRarefairlyinvolvedandcomplex.Themathinvolvedisabovethescopeofthiswebsite,butthiswebsiteexplainssomespecificsnottalkedabouthere:
www.wzw.tum.de.
Thelightemittedfromthedyeintheexcitedstateisreceivedbyacomputerandshownonagraphdisplay,suchasthis,showingPCRcyclesontheX-axisandalogarithmicindicationofintensityontheY-axis.
Figure4.AgraphprintioutofactualdatafoundusingtheTaqMan®
probe.Courtesywww.biotech.uiuc.edu.
Figure5.Areal-timePCRmachineusedatColoradoState.Courtesylamar.colostate.edu.
OtherImagesofTaqMan®
inAction:
Figure6.AnotherthreestepviewoftheTaqMan®
probeworking:
beforetheprobeismetwiththeTaqpolymerase,energyistransferredfromashort-wavelengthfluorophore(green)toalong-wavelengthfluorophore(red).Whenthepolymeraseaddsnucleotidestothetemplatestrand,itreleasestheshort-wavelengthfluorophore,makingitdetectableandthelong-wavelengthundetectable.Figurecourtesy
Figure7.AnotherviewofTaqMan®
inaction.ThereleasefromtheQuencherdye(redQ)instep2eventuallycausestheReporterdye(blueR)tobeseeninstep4.Figurecourtestywww.ruhr-uni-bochum.depending.
OtherReal-TimePCRMethods:
Therearetwoothertypesofreal-timePCRmethods,themolecularbeaconmethodandtheSYBR®
Greenmethod.Themolecularbeaconmethodutilizesareporterprobethatiswrappedaroundintoahairpin.Italsohasaquencherdyethatmustbeinclosecontacttothereportertowork.AnimportantdifferenceofthemolecularbeaconmethodincomparisontotheTaqMan®
methodisthattheproberemainsintactthroughoutthePCRproduct,andisreboundtothetargetateverycycle.ClickheretoseeawebpageonthemolecularbeaconmethodofPCR,anothertypeofreal-timePCRusedinmolecularbiology.TheSYBR®
Greenprobewasthefirsttobeusedinreal-timePCR.Itbindstodouble-strandedDNAandemitslightwhenexcited.Unfortunately,itbindstoanydouble-strandedDNAwhichcouldresultininaccuratedata,especiallycomparedwiththespecificityfoundintheothertwomethods.(2003)
References:
Campbell,Malcolm.PCRmovie.<
http:
//www.bio.davidson.edu/misc/movies/pcr2.mov>
Accessed2/17/03.
Campbell,Malcolm.Real-timePCR,molecularbeaconmethod..<
//www.bio.davidson.edu/courses/genomics/method/realtimepcr.html>
ColoradoStateLaboratoryhomepage.<
//lamar.colostate.edu/~reddy/labtour2.htm>
FluorescenceResonanceEnergyTransfer(FRET).<
Accessed2/18/03.
HistoryofPCR.<
//usitweb.shef.ac.uk/~mba97cmh/history/history.htm>
Accessed2/15/03.
Purves,etal.Life:
TheScienceofBiology.SixthEdition.FreemanandCompany:
USA;
2001.pg.214-217.
"
Real-Time"
orKineticPCR.<
//dna-9.int-med.uiowa.edu/realtime.htm>
Real-timeRT-PCR.<
W.M.KeckCenterforComparitiveandFunctionalGenomics.<
//www.biotech.uiuc.edu/taqman.htm>
GeneQuantification.<
//www.wzw.tum.de/gene-quantification/relative.html>
Accessed2/16/03.