GeneralAdvice

PCRallowstheproductionofmorethan10millioncopiesofatargetDNAsequencefromonlyafewmolecules.ThesensitivityofthistechniquemeansthatthesampleshouldnotbecontaminatedwithanyotherDNAorpreviouslyamplifiedproducts(amplicons)thatmayresideinthelaboratoryenvironment.

GuidanceinAvoidingContamination

• DNAsamplepreparation,reactionmixtureassemblageandthePCRprocess,inadditiontothesubsequentreactionproductanalysis,shouldbeperformedinseparateareas.
• ALaminarFlowCABInetequippedwithaUVlampisrecommendedforpreparingthereactionmixture.
• FreshglovesshouldbewornforeachPCRstep.
• TheuseofdedicatedvesselsandpositivedisplacementPipettesortipswithaerosolfiltersforbothDNAsampleandreactionmixturepreparation,isstronglyrecommended.
• ThereagentsforPCRshouldbepreparedseparatelyandusedsolelyforthispurpose.Autoclavingofallsolutions,exceptdNTPs,primersandPfuDNAPolymeraseisrecommended.SolutionsshouldbealiquotedinsmallportionsandstoredindesignatedPCRareas.AliquotsshouldbestoredseparatelyfromotherDNAsamples.
• Acontrolreaction,omittingtemplateDNA,shouldalwaysbeperformed,toconfirmtheabsenceofcontamination.

Theseareonlyroughguidelines.DetailedinstructionsaboutPCRlaboratorysetupandmaintenancemaybefoundinPCRMethodsandApplications,3,2,S1-S14,1993.

PreparationofReactionMixture

Toperformseveralparallelreactions,werecommendthepreparationofamastermixcontainingwater,buffer,dNTPs,primersandtemplateDNAinasingletube,whichcanthenbealiquotedintoindividualtubes.PfuDNAPolymeraseshouldbeaddedlast.Thismethodofsettingreactionsminimizesthepossibilityofpipettingerrorsandsavestimebyreducingthenumberofreagenttransfers.

ReactionMixtureSetUp

• Gentlyvortexandbrieflycentrifugeallsolutionsafterthawing.
• Keepsolutionsonice.
• Add,inathin-walledPCRtube,onice:

Reagent	Finalconcentration	Quantity,for50µlofreactionmixture
Steriledeionizedwater	-	variable
10XPCRbufferwithMgSO4*	1X	5µl
2mMdNTPmix	0.2mMofeach	5µl
PrimerI	0.1-1µM	variable
PrimerII	0.1-1µM	variable
TemplateDNA	50pg-1µg	variable
PfuDNAPolymerase	1.25u/50µl	0.5µl

*Ifusing10XPCRbufferwithoutpre-addedMgSO₄,refertothetablebelowtodeterminerequiredvolumeof25mMMgSO₄solution(for50µltotalvolume).

FinalconcentrationofMgSO4in50µlreactionmix,mM	1.0	1.25	1.5	1.75	2.0	2.5	3.0	4.0
Volumeof25mMMgSO4,µl	2	2.5	3	3.5	4	5	6	8

• Gentlyvortexthesampleandbrieflycentrifugetocollectalldropsfromwallsoftube.
• Ifusingathermalcyclerwithoutaheatedlid,overlaythesamplewithahalfvolumeofmineraloiloraddanappropriateamountofwax.
• Placesamplesinathermalcyclerpreheatedto95°CandstartPCR.

Recommendedthermalcyclingconditions:

Step	Temperature,°C	Time,min	Numberofcycles
Initialdenaturation	95	1-3	1
Denaturation	95	0.5-2
Annealing	37-70	0.5-2	25-35
Extension	70-75	2-4
Finalextension	70-75	5	1

Note

CompositionoftheReactionMixture

1. TemplateDNA.UsuallythetemplateDNAamountisintherangeof50pg-1ngforplasmidorphageDNAand0.1-1µgforgenomicDNA,foratotalreactionmixtureof50µl.HighertemplateDNAamountsusuallyincreasetheyieldofnonspecificPCRproducts,butifthefidelityofsynthesisiscrucial,maximalallowabletemplateDNAquantitiesinconjunctionwithlimitingnumberofPCRcyclesshouldbeusedtoincreasethepercentageof"correct"PCRproducts.NearlyallroutinemethodsaresuitablefortemplateDNApurification.AlthougheventraceamountsofagentsusedinDNApurificationprocedures(phenol,EDTA,ProteinaseK,etc.)stronglyinhibitPfuDNAPolymerase,ethanolprecipitationofDNAandrepetitivetreatmentsofDNApelletswith70%ethanolisusuallyeffectiveinremovingtracesofcontaminantsfromtheDNAsample.
2. Primers.Guidelinesforprimerselection:
   • PCRprimersareusually20-30nucleotidesinlength.Longerprimersprovidesufficientspecificity.
   • TheGCcontentshouldbe40-60%.TheCandGnucleotidesshouldbedistributeduniformlywithinthefulllengthoftheprimer.MorethanthreeGorCnucleotidesatthe3-endoftheprimershouldbeavoided,asnonspecificprimingmayoccur.
   • Theprimershouldnotbeself-complementaryorcomplementarytoanyotherprimerinthereactionmixture,inordertoavoidprimer-dimerandhairpinformation.
   • Themeltingtemperatureofflankingprimersshouldnotdifferbymorethan5°C,sotheGCcontentandlengthmustbechosenaccordingly.
   • AllpossIBLesitesofcomplementaritybetweenprimersandthetemplateDNAshouldbenoted.
   • Ifprimersaredegenerate,atleast3conservativenucleotidesmustbelocatedattheprimers3-end.
   • Estimationofthemeltingandannealingtemperaturesofprimer:Iftheprimerisshorterthan25nucleotides,theapprox.meltingtemperature(T_m)iscalculatedusingthefollowingformula:T_m=4(G+C)+2(A+T)G,C,A,T-numberofrespectivenucleotidesintheprimer.Iftheprimerislongerthan25nucleotides,themeltingtemperatureshouldbecalculatedusingspecializedcomputerprogramswheretheinteractionsofadjacentbases,theinfluenceofsaltconcentration,etc.areevaluated.Optimalannealingtemperatureisgenerally5°Clowerthanthemeltingtemperatureoftheprimer-templateDNAduplex.
   • The3’=>5’exonucleaseactivityassociatedwithPfuDNAPolymerasemaydegradetheprimers.ItisthereforeimportantthatPfuDNAPolymerasebeaddedlasttothereactionmixture.Degradationofprimerscanbeefficientlypreventedbytheintroductionofphosphorothioatebondsattheir3’-termini(1).TheuseoflongerprimerswithmaximizedGCcontentcanbeadvantageous.
3. MgSO₄concentration.PfuDNAPolymeraseprefersMgSO₄toMgCl₂.SinceMg²⁺ionsformcomplexeswithdNTPs,primersandDNAtemplates,theoptimalconcentrationofMgSO₄hastobeselectedforeachexperiment.ToofewMg²⁺ionsresultinalowyieldofPCRproduct,andtoomanyincreasetheyieldofnon-specificproducts.OptimalMgSO₄concentrationisintherangeof2-4mM.IncreasingMgSO₄concentrationfrom2to10mMdidnotvarysignificantlytheerrorrate.IftheDNAsamplescontainEDTAorotherchelators,theMgSO₄concentrationshouldberaisedproportionally.
4. dNTPs.TheconcentrationofeachdNTPinthereactionmixtureisusually200µM.ItisveryimportanttohaveequalconcentrationsofeachdNTP(dATP,dCTP,dGTP,dTTP),asinaccuracyintheconcentrationofevenasingledNTPdramaticallyincreasesthemisincorporationlevel.dNTPsconcentrationsof100-250µMofeachdNTPresultintheoptimalbalanceofproductyield(greaterathigherdNTPconcentration)versusspecificity.Inaddition,theoptimalconcentrationofdNTPsshouldbeselectedempirically.
5. PfuDNAPolymerase.
   • TheconcentrationoftheenzymerequiredforoptimalPCRproductyieldandspecificitydependontargettobeamplifiedandthepresenceofinhibitorsinthereactionmix(e.g.,ifthetemplateDNAusedisnothighlypurified).Theoptimalenzymeconcentrationis1.25-2.5u/50µl.
   • 3’=>5’exonucleaseactivityassociatedwithPfuDNAPolymerasemaydegradeprimers.ItisimportanttoaddtheenzymetothereactionmixtureatlastandplacePCRmixesandtubesonice.
   • PfuDNAPolymerasehasnodetectablereversetranscriptaseactivity.
6. Reactionoverlay.Ifnecessary,thereactionmixturecanbeoverlaidwithmineraloilorparaffin(meltingtemperature50-60°C)ofspecialPCRgrade.One-halfofthetotalreactionvolumeisusuallysufficient.

TemperatureCycling

Amplificationparametersdependgreatlyonthetemplate,primersandamplificationapparatusused.

1. InitialDenaturationStep.
   • ThecompletedenaturationoftheDNAtemplateatthestartofthePCRreactionisofkeyimportance.IncompletedenaturationofDNAresultsintheinefficientutilizationoftemplateinthefirstamplificationcycleandinapooryieldofPCRproduct.Theinitialdenaturationshouldbeperformedoveranintervalof1-3minat95°CiftheGCcontentis50%orless.Thisintervalshouldbeextendedupto10minforGC-richtemplatesordenaturationtemperaturemaybeincreasedupto97°C.
2. DenaturationStep.
   • Usually0.5-2mindenaturationat94-95°Cissufficient,sincethePCRproductsynthesizedinthefirstamplificationcycleissignificantlyshorterthanthetemplateDNAandiscompletelydenaturedundertheseconditions.TheGCcontentshouldbetakenintoconsideration.Denaturationtimecanbeoptimizedempirically.
3. PrimerAnnealingStep.
   • Usuallytheoptimalannealingtemperatureis5°Clowerthanthemeltingtemperatureofprimer-templateDNAduplex.Incubationfor0.5-2minisusuallysufficient.However,ifnon-specificPCRproductsareobtainedinadditiontotheexpectedproduct,theannealingtemperatureshouldbeoptimizedbyincreasingitstepwiseby1-2°C.
4. ExtendingStep.
   • Usuallytheextendingstepisperformedat70-75°C.PfuDNAPolymeraseexhibitslowerthanthatofTaqDNAPolymeraseextentionrate(0.5kb/min),so2minextentiontimeisrecommendedforevery1kbtobeamplified.
5. NumberofCycles.
   • ThenumberofPCRcyclesdependsontheamountoftemplateDNAinthereactionmixandontheexpectedyieldofthePCRproduct.Formostamplificationreactions,25-35cyclesareusuallysufficient.Ingeneral,wesuggestusingthefewestcyclespossibletoachieveacceptableyieldofPCRproductandensurelessamountofnon-specificbackgroundproduct.
6. FinalExtendingStep.
   • Afterthelastcycle,thesamplesareusuallyincubatedat72°Cfor5-15mintofill-intheprotrudingendsofnewlysynthesizedPCRproducts.
   • PfuDNAPolymerase,unlikeTaqDNAPolymerase,lacksterminaltransferaseactivityandgeneratesblunt-endedPCRproducts,whichcanbeuseddirectlyforbluntendligationwithoutanypretreatmentoftheends.

Reference

1. Skerra,A.,PhosphorothioateprimersimprovetheamplificationofDNAsequencesbyDNApolymeraseswithproofreADIngactivity,NucleicAcidsRes.,20,3551-3554,1992.

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