RealTime PCR.docx

RealTime PCR.docx

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RealTimePCR

Real-TimePCR:

theTaqMan®Method

ThiswebpagewasproducedasanassignmentforanundergraduatecourseatDavidsonCollege.

Introduction:

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（orFörster）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.

//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>Accessed2/17/03.

ColoradoStateLaboratoryhomepage.

//lamar.colostate.edu/~reddy/labtour2.htm>Accessed2/17/03.

FluorescenceResonanceEnergyTransfer（FRET）.

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>Accessed2/17/03.

Real-timeRT-PCR.

W.M.KeckCenterforComparitiveandFunctionalGenomics.

//www.biotech.uiuc.edu/taqman.htm>Accessed2/17/03.

GeneQuantification.

//www.wzw.tum.de/gene-quantification/relative.html>Accessed2/16/03.