Thestepsof:

1. Templatedenaturation
2. Primerannealing
3. Primerextension

AftereachcyclethenewlysynthesizedDNAstrandscanserveastemplatesinthenextcycle.

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)

(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:

1. Competitionofcomplementarydaughterstrandswithprimersforreannealing(i.e.twodaughterstrandsreannealingresultsinnoamplification).
2. Lossofenzymeactivityduetothermaldenaturation,especiallyinthelatercycles
3. Evenwithoutthermaldenaturation,theamountofenzymebecomeslimitingduetomolartargetexcessinlatercycles(i.e.after25-30cyclestoomanyprimersneedextending)
4. Possiblesecondsiteprimerannealingandnon-productivepriming

PCRwasinventedin1985byKaryMullis,workingforCetuscorporationsomewherenearBerkeley,California.TheoriginalmethodofPCRusedtheKlenowfragmentofE.coliDNApolymeraseI.Thisenzyme,however,denaturesattemperatureslowerthanthatrequiredtodenaturemosttemplateduplexes.Thus,aftereachcycle,freshenzymehadtobeaddedtothereaction.Thiswasquitetedious.Inadditiontothisproblemwiththeenzyme,thesampleshadtobemovedfromonetemperaturebathtoanothertoallowtheindividualstepsofdenaturation,annealingandpolymerization(whichallrequireddifferenttemperatures.Thiswasprettytedioustoo.

• Thus,twomainadvancesallowedtheprocesstobeautomated,theseadvanceswere:

1. TheuseofthermostableDNApolymerases,whichresisteddenaturation(inactivation)athightemperatures.Thus,aninitialaliquotofpolymerasecouldlastthroughoutthenumerouscyclesoftheprotocol.ThefirstthermostableDNApolymerasetobeusedwasisolatedfromthebacteriumThermusaquaticus.ItwasisolatedfromahotspringinYellowstoneNationalParkwhereitlivedhappily(i.e.itreplicateditsDNA)attemperaturesinexcessof85°C
2. Thedevelopmentoftemperaturebathswhichcouldshifttheirtemperaturesupanddownrapidlyandinanautomatedprogrammedmanner.TheseareknownasthermalcyclersorPCRmachines.

ThesetwodevelopmentslettotheautomationofPCR.ThePCRprocessiscoveredbypatentsownedbyHoffmann-LaRocheInc.(afacelessconglomerateyoucantrust).