PCR.docx
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PCR.docx
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PCR
AmplificationofDNAfragmentsbyPolymeraseChainReaction(PCR)andtheAnalysisofPCRProducts
Ⅰ、ObjectivesoftheExperiment:
(1)LearntheprincipleandmethodofThePolymeraseChainReaction(PCR),knowabouttailPCR.
(2)ComprehendthesignificanceofPCRtechniqueinDNAmanipulation.
Ⅱ、ThePrincipleoftheexperiment
(1)BackgroundsandConcepts
Thepolymerasechainreaction(PCR)isatechniqueforamplifyingDNAsequencesinvitro.PCRwasinventedbyKaryMullisandhiscolleaguesin1985.ThediscoveryofTaqDNApolymerase,thermallystableenzymeisolatedbyChienetal.in1976,madethePCRautomationpossible.ThismethodtakesadvantageofthermallystableDNApolymeraseandcanproducenumerouscopiesfromasingletemplateDNAmoleculethroughtensofrepeatedcyclesoftemplatedenaturation,primerannealing,andDNAsynthesis,butitisextremelysensitivetotracecontaminationofunwantedtemplateDNAinthereactionsolution.What’smore,thismethodisalsoimportantinbiotechnology,forensicidentification,medicine,andgeneticresearch.
(2)Principlesofthewholeexperiment
1.PrincipleofPCR:
SimilartoDNAdenaturationandrenaturationathightemperature(93-95℃),thetargetdouble-strandDNAcanbeseparatedintosingle-strandDNA.Atlowtemperature(37-65℃),twoartificialoligonucleotideswillannealedtothecomplementarysequenceinthetemplateformingpartialdoublestrand.At72℃,theTaqpolymerasesynthesizesnewstrandbyextendingtheprimersalongthedirectionfrom5’to3’.Thenumberofsequencesbetweentheprimersisdoubledafterthiscycle.ThecyclecanberepeatedasthenewlysynthesizedDNAstrandscanserveastemplatesinthenextcycle.So,theamountoftargetsequencedramaticallyincreases.Theamplificationcoefficientcanbeabove109theoreticallyafter25-30cyclesor106-107practically.
2.PCRReactionMixture:
DNAtemplate(purifiedoracrudeextract):
PCRcanbeperformedusingssDNA,dsDNAorcDNA(reversetranscribedfromRNA)astemplate.Thelinearizedplasmidispreferredwhenplasmidservesastemplate.However,thecircularplasmidcanbealsousedastemplatedirectlyinmostcases.Digestionwithappropriateenzymemaygivebetteramplificationresultwhenthetemplateislong(e.g.genomicDNA).
Attemplatedenaturatingtemperature,thedouble-strandDNAmeltsandopensintosingle-strandDNA.Recommendeddenaturatingtemperaturerangesfrom90to95oC.ThecompletedenaturationoftheDNAtemplateatthestartofthePCRreactionisofkeyimportance.IncompletedenaturationofDNAresultsintheinefficientutilizationoftemplateinthefirstamplificationcycleandinapooryieldofPCRproduct.DNAdenaturationrequiresonlyafewseconds.Thedenaturatingtimeshouldbeasshortaspossibleinordertoremainthepolymeraseactivity.Thedenaturatingtemperatureshouldnotbeover95oC,asthestabilityoftheenzymedramaticallydecreasesattemperatureover95°C.
PrimersspecificforthetargetDNA:
Twoprimersshouldbedesignedbeforeamplification.TheyarecomplementarytothebothendsoftargetDNAsequencerespectively.Thesetwoprimersdeterminethelengthandlocusoftheamplifiedfragment.So,thedesignofprimerisveryimportant.Thenumberofnucleotidesinaprimerisusually16-30nt.Apreferablenumberis20-24nt.Sometimesrestrictionsitesandenhancerfactors,whicharenotcomplementarytotemplate,canbeaddedtoprimer5’endsinordertoaccomplishgenecloningandotherspecialtasks.Underthiscondition,additional3-4nucleotidesasflankingsequencespacershouldbeaddedto5’endforefficientdigestion.
Primerconcentrationbetween0.1and1Misgenerallyoptimal.Higherprimerconcentrationmayeasilygenerateprimer-dimerandnonspecificproduct.Lowerprimerconcentrationmayresultinhigherspecificity,buttoolowconcentrationisnotsufficienttoaccomplish30amplificationcyclesandwilldecreasethePCRyield.
PrimerannealingtemperaturedeterminesthePCRspecificityandyield.Higherannealingtemperatureshouldresultinenhancedspecificitywhiletoohighannealingtemperaturemayaffecttheassociationofprimerswithtemplateanddecreasetheamplificationefficiency.DecreasingannealingtemperaturecanincreasetheamountofPCRproduct,buttoolowannealingtemperaturemayinducemispairingandincreasenonspecificproducts.
Optimalannealingtemperatureisgenerally5oClowerthanthemeltingtemperature(Tm)oftheprimer-templateDNAduplex.TheapproximateTmcanbecalculatedusingthefollowingformula:
Tm=4(G+C)+2(A+T)
G,C,A,T-numberofrespectivenucleotidesintheprimer.Attypicalprimerconcentrations(0.2μM),annealingwillrequireonlyafewseconds.
Primer1(FrompCMV-Myc827-847):
5’TTCCAAGCTTCACCATGGCATCAATGCAGAAGC
5’ClampHindIIIMyctag
Tm=4(G+C)+2(A+T)=4×12+2×11=700C.
Primer2(FromT10AK076127.1|1249-1270):
5’TCGCGGATCCAGTGGCATCAGAGACTTGCTAATC5’ClampBamHI
Tm=4(G+C)+2(A+T)=4×11+2×13=700C.
FreeDeoxynucleotideTriphosphates(dNTPs):
InPCRreactionsystem,higherthan50mMdNTPinhibitsTaqactivity.ExcessivedNTPconcentrationmayincreasetheerrorrate.LoweringthedNTP(10-50uM)mayreduceerrorrate,buttoolowconcentrationwillnotproduceenoughamountofPCRproducts.dNTPsontherangeof50-200Maregenerallyappropriate.10~15Misminimumconcentration.FourdNTPs(dATP,dTTP,dGTP,dCTP)areusedatequivalentconcentrationstominimizemis-incorporation.
TaqDNApolymerase:
TaqwasisolatedfromThermusaquaticus,bacteriathatgrowsinhotsprings(75℃).AtypicalPCRreactionrequires2Uenzyme.Thecommonrangeis1~4U/100l.Theenzymerequirementsmayvarywithrespecttoindividualtargettemplatesorprimers.Excessivepolymerasemayincreasetheamountofnon-specificPCRproducts.Oneunitistheamountoftheenzymethatwillincorporate10nmolofdNTPintoacid-insolubleproductsin30minutesat74oCwithactivatedsalmonspermDNAasthetemplate-primer.
Buffer(containingMg2+):
AstandardbufferforPCRis10-50mMTris-HCl(pH8.3)and1.5mMMgCl2.At72℃,thereactionsystempHdecreases1unit,closing7.2.ThedivalentcationisessentialwhichmayaffectPCRspecificityandproductamount.Mg2+isbetterthanMn2+,whileCa2+hasnoeffect.Reducedconcentrationcanpreventnon-specificandundesirablePCRproducts.Increasedconcentrationcanattainmoreproduct.TheconcentrationofMg2+shouldbeoptimizedatfirsttimeofassociationoftargetsequenceandprimers.Itiswithinarangeof1-10mMgenerally.
3.PCRAmplificationProcess
ThreemajorstepsareinvolvedinaPCRcycle.Thecyclesaredoneonanautomatedcycler,whichrapidlyheatsandcoolsthetesttubescontainingthereactionmixture.Eachcyclecomprisesthefollowingthreesteps:
1.Denaturation:
Athightemperature(90-95oC),thedouble-strandDNAmeltsandopensintosingle-strandDNA.
2.Annealing:
Atlowtemperature(35-65oC),single-strandprimerbindstothesingle-strandtemplateformingpartialdoublestrand.
3.Extension:
At72oC,theDNApolymeraseextendstheprimersbyreadingtheopposingstrandsequenceandaddingnucleotides.Thenumberoftargetsequencesisdoubledaftereachcycle.
Figure1DNAAmplificationUsingPolymeraseChainReaction
Ⅲ、Instruments,MaterialsandReagents
1、Instruments
PCRsystem,centrifuge,Eppendorftubes,gelelectrophoresissystem,UVGelimaginginstruments
2、MaterialsandReagents
Chart1.1MaterialandReagentsofPCR
MaterialsandReagents
Quantity
TaKaRaTaq
5U/l
dNTPMixture
2.5mM
Template
10ng/l
Primer1
1M
Primer2
1M
10XPCRBuffer
100mMTris-HCl,pH8.3,500mMKCl,15mMMgCl2
TAEBuffer(10X)
LoadingBuffer(3X)
0.25%Bromophenolblue,40%(W/V)sucroseor30%glycerol
EB
10mg/ml
ddH2O
Ⅳ、Procedures
I.TaketheEppendorftubeandaddthematerialsandreagentsintothetube,theingredientsareshowninChart1.2below.
Chart1.2theIngredientsofthePCRMixture
Ingredients
VolumeandQuantity
TemplateDNA
1l(10ng/l)
Primer1
10l(1M)
Primer2
10l(1M)
dNTPs
4l(2.5mM)
TaqDNApolymerase
0.5l(5U/l)
10×PCRBuffer(Mg+)
5l
ddH2O
19.5l
Total
50l
II.PutthetubeintotheautomaticPCRapparatus;automaticallyproceedfor30Cycles.(DetailedProcessareshowninChart1.3below)
Chart1.3thePCRCycles
SerialNumber
Procedures
1
Denaturationat94℃for5minutes
2
Denaturationat94℃for45seconds
3
Annealingat65℃for45seconds
4
Extensionat72℃for45seconds
5
Repeatprocedure2to4for30times
6
Finalextensionat72℃for7minutes
7
Chillingto4℃
III.AnalyzetheproductbytheGelElectrophoresis.
ThePCRproductamountandspecificityareanalyzedbyethidiumbromide-stained1.0%agarosegelelectrophoresisusing4lPCRreactionmixture.TheamountofPCRproductcanbeestimatedbycomparisonwithstandardDNAladderapproximately.
1.PreparetheAgaroseGel;
2.Addthe1XTAEBufferintotheGelElectrophoresisApparatus;
3.MixtheproductofPCRwiththeloadingbuffer(4lPCRproductand8lLoadingBuffer);
4.LoadthesampleintheAgarosegel(12lforeachwell);
5.Add1kbNEBDNAladder(50ng/μl)tothecertainwells;
6.Starttheelectrophoresisofthegelintheelectrophoresisapparatus(thepressureisabout90V);
7.GelElectrophoresisforabout40minutes;
8.TakeouttheAgarosegelanduseEBtodyethegelfor10minutes;
9.handletheUVimagingsystemtogettheUVGelElectrophoresisImage.
IV.CollectandPurifytheproducts
1.AdjustthevolumeofthePCRsampleto50lwithwaterifnecessary.Add8volumesofBindingBufferto1volumeofthePCRsampleandmix.
Attention:
Forastandardpurification,add400μlofBindingBufferto50μlPCR
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