PolymeraseChainReaction(PCR)
- PCRisaninvitrotechniquefortheamplificationofaregionofDNAwhichliesbetweentworegionsofknownsequence.
- PCRamplificationisachievedbyusingoligonucleotideprimers.
- Thesearetypicallyshort,singlestrandedoligonucleotideswhicharecomplementarytotheouterregionsofknownsequence.
- TheoligonucleotidesserveasprimersforDNApolymeraseandthedenaturedstrandsofthelargeDNAfragmentservesasthetemplate.
- ThisresultsinthesynthesisofnewDNAstrandswhicharecomplementarytotheparenttemplatestrands.
- Thesenewstrandshavedefined5"ends(the5"endsoftheoligonucleotideprimers),whereasthe3"endsarepotentiallyambiguousinlength.
- TheoligonucleotidedirectedsynthesisofdaughterDNAstrandscanberepeatedifthenewduplexisdenatured(byheating)andadditionalprimersareallowedtoanneal(bycoolingtoanappropriatetemperature).
Thestepsof:
- Templatedenaturation
- Primerannealing
- Primerextension
compriseasingle"cycle"inthePCRamplificationmethodology.
AftereachcyclethenewlysynthesizedDNAstrandscanserveastemplatesinthenextcycle.
Notethathalfofthenewlysynthesizedstrandsfromthesecondroundofreplicationhave5"and3"terminiwhicharedefinedbytheannealinglocationoftheprimeroligonucleotides.
SummaryofproductsattheendofeachPCRcycle:
- Thetotalofeachofthedifferenttypesofoligonucleotidefragmentsforeachcyclecanbesummarizedasfollows:
- Theamplificationofthefragmentsfollowsthefollowingpattern:
- Thereisalways1xcopyofeachoftheoriginaltemplates-thePCRasoutlinedneverreproducesthefulllengthtemplate
- Therewillbe"n"xcopiesofeachfragmentwithanindeterminatelength(where"n"isthenumberofcycles).ThefragmentsofindeterminantlengthhaveoneenddefinedbyaPCRprimerandtheotherendisofindeterminantlength
- Therewillbe(2n-(n+1))xcopiesofeachfragmentofdefinedlength(i.e.eachenddefinedbythetwoPCRprimers).
Forexample,after6cycleswewillhave- 1xcopyofeachoriginaltemplate
- 6xcopiesofeachfragmentofindeterminantlength
- (64-(6+1))=57xcopiesofeachfragmentofdefinedlength
Intheabovecase,thedesiredPCRproductwillbeaduplexofthedefinedlengthfragment.Thequestionis:howmanywillbeproduced?- Theoriginaltemplateswillnotnecessarilyannealwithoneanother
- Likewise,thetemplatesofindeterminantlengthwillnotnecessarilyannealwithoneanother
- FurThermore,asthenumberofcyclesproceeds,thedefinedlengthfragmentsfaroutnumbertheothers
- Therefore,theoriginaltemplate,andfragmentsofindeterminantlengtharemostlikelygoingtohybridizewithdefinedlengthfragments
- Ourexpectedamplificationwouldthereforebe(usingthe6cyclePCRexample)
57-(6+1)=50xcopiesofeachdefinedlengthfragments
(i.e.50xduplexesofdefinedlength)
Theexpectedamplificationofthedesireddefinedlengthproductwithrespecttotheoriginaltemplateconcentration"x"canthusberepresentedbytheformula:[(2n-(n+1))-(n+1)]x
or
(2n-2(n+1))
(thisisoftenabbreviatedtoasimpleruleofthumbfortheamplification:(2n-2n)x)- Theinterpretationofthisformulaisthat
- Foragivennumberofcycles"n"wemake"2nx"totalpossIBLeduplexes
- Foragivennumberofcyclestherewillbe"2(n+1)(or2ninourapproximation)x"duplexeswhichareformedfromeithertheoriginaltemplate,orafragmentofindeterminatelength,alongwithafragmentofdefinedlength(andrepresentanundesiredproduct)
- Thus,thetotalconcentrationofdesiredproduct(duplexeswithalengthdefinedbythePCRprimers)willbe(2n-2(n+1))x(wherexistheconcentrationoftheoriginalduplex)
Thetheoreticalamplificationvalueisneverachievedinpractice.Severalfactorspreventthisfromoccuring,including:- Competitionofcomplementarydaughterstrandswithprimersforreannealing(i.e.twodaughterstrandsreannealingresultsinnoamplification).
- Lossofenzymeactivityduetothermaldenaturation,especiallyinthelatercycles
- Evenwithoutthermaldenaturation,theamountofenzymebecomeslimitingduetomolartargetexcessinlatercycles(i.e.after25-30cyclestoomanyprimersneedextending)
- Possiblesecondsiteprimerannealingandnon-productivepriming
PCRwasinventedin1985byKaryMullis,workingforCetuscorporationsomewherenearBerkeley,California.TheoriginalmethodofPCRusedtheKlenowfragmentofE.coliDNApolymeraseI.Thisenzyme,however,denaturesattemperatureslowerthanthatrequiredtodenaturemosttemplateduplexes.Thus,aftereachcycle,freshenzymehadtobeaddedtothereaction.Thiswasquitetedious.Inadditiontothisproblemwiththeenzyme,thesampleshadtobemovedfromonetemperaturebathtoanothertoallowtheindividualstepsofdenaturation,annealingandpolymerization(whichallrequireddifferenttemperatures.Thiswasprettytedioustoo.
- Thus,twomainadvancesallowedtheprocesstobeautomated,theseadvanceswere:
- TheuseofthermostableDNApolymerases,whichresisteddenaturation(inactivation)athightemperatures.Thus,aninitialaliquotofpolymerasecouldlastthroughoutthenumerouscyclesoftheprotocol.ThefirstthermostableDNApolymerasetobeusedwasisolatedfromthebacteriumThermusaquaticus.ItwasisolatedfromahotspringinYellowstoneNationalParkwhereitlivedhappily(i.e.itreplicateditsDNA)attemperaturesinexcessof85°C
- Thedevelopmentoftemperaturebathswhichcouldshifttheirtemperaturesupanddownrapidlyandinanautomatedprogrammedmanner.TheseareknownasthermalcyclersorPCRmachines.
ThesetwodevelopmentslettotheautomationofPCR.ThePCRprocessiscoveredbypatentsownedbyHoffmann-LaRocheInc.(afacelessconglomerateyoucantrust).
Thermalcyclingparameters
ThethermalcyclingparametersarecriticaltoasuccessfulPCRexperiment.TheimportantstepsineachcyclesofPCRinclude:- denaturationoftemplate
- annealingofprimers
- extensionoftheprimers
Arepresentativetemperatureprofileforeachcyclemightlooklikethefollowing:
Templatedenaturation
Theinitialdenaturationoftemplateisaccomplishedat95-100°C.- Supercoiledplasmidsaretoughertomeltandmayrequireboilingforseveralminutes,ormaybeinitiallydenaturedbyusingbase(NaOH,followedbypHneutralization).
- DenaturationduringthePCRexperiment(i.e.secondcycleonward)isusuallyaccomplishedattemperaturesof92-95°C(usuallyempiricallydetermined).
Primerannealingtemperature
PrimerannealingtemperatureisanimportantparameterinthesuccessofthePCRexperiment.Theannealingtemperatureischaracteristicforeacholigonucleotide:- itisafunctionofthelengthandbasecompositionoftheprimeraswellastheionicstrengthofthereactionbuffer.
- Estimatesoftheannealingtemperaturecanbecalculatedinseveraldifferentways.
- ThesecalculatedannealingtemperaturesareastartingpointforthePCRexperiment,butidealannealingtemperaturesaredeterminedempirically.
Primerextension
Primerextensionisusuallyperformedat72°C,ortheoptimumtemperatureoftheDNApolymerase.ThelengthoftimeoftheprimerextensionstepscanbeincreasediftheregionofDNAtobeamplifiedislong,however,forthemajorityofPCRexperimentsanextensiontimeof2minutesissufficienttogetcompleteextension.Numberofcycles
Thenumberofcyclesisusuallybetween25and35.- Morecyclesmeanagreateryieldofproduct.
- However,withincreasingnumberofcyclesthegreatertheprobABIlityofgeneratingvariousartifacts(e.g.misprimingproducts).
- Itisunusualtofindprocedureswhichhavemorethan40cycles.
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