核酸恒温扩增技术HDAWord文档下载推荐.docx
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Copyright©
2004,EuropeanMolecularBiologyOrganization
ScientificReport
Helicase-dependentisothermalDNAamplification
MyriamVincent,1*YanXu,1*andHuiminKong1a
1NewEnglandBiolabs,32TozerRoad,Beverly,Massachusetts01915,USA
aTel:
+19789275054;
Fax:
+19789211350;
E-mail:
kong@
*Theseauthorscontributedequallytothiswork
ReceivedJanuary14,2004;
RevisedMay24,2004;
AcceptedJune14,2004.
ThisarticlehasbeencitedbyotherarticlesinPMC.
∙
OtherSections▼
oAbstract
oIntroduction
oResults
oDiscussion
oMethods
oSupplementaryMaterial
oReferences
Abstract
PolymerasechainreactionisthemostwidelyusedmethodforinvitroDNAamplification.However,itrequiresthermocyclingtoseparatetwoDNAstrands.Invivo,DNAisreplicatedbyDNApolymeraseswithvariousaccessoryproteins,includingaDNAhelicasethatactstoseparateduplexDNA.WehavedevisedanewinvitroisothermalDNAamplificationmethodbymimickingthisinvivomechanism.Helicase-dependentamplification(HDA)utilizesaDNAhelicasetogeneratesingle-strandedtemplatesforprimerhybridizationandsubsequentprimerextensionbyaDNApolymerase.HDAdoesnotrequirethermocycling.Inaddition,itoffersseveraladvantagesoverotherisothermalDNAamplificationmethodsbyhavingasimplereactionschemeandbeingatrueisothermalreactionthatcanbeperformedatonetemperaturefortheentireprocess.ThesepropertiesofferagreatpotentialforthedevelopmentofsimpleportableDNAdiagnosticdevicestobeusedinthefieldandatthepoint-of-care.
Keywords:
DNAamplification,isothermal,helicase,DNApolymerase,UvrD
Introduction
Thepolymerasechainreaction(PCR)revolutionizedourcapabilitiestodobiologicalresearch,andithasbeenwidelyusedinbiomedicalresearchanddiseasediagnostics(Saikietal,1988).Hand-helddiagnosticdevices,whichcanbeusedtodetectpathogensinthefieldandatpoint-of-care,aredemandedcurrently.However,theneedforpower-hungrythermocyclinglimitsPCRapplicationinsuchasituation.Severalisothermaltargetamplificationmethodshavebeendeveloped(Andrasetal,2001).Strand-displacementamplification(SDA)combinestheabilityofarestrictionendonucleasetonicktheunmodifiedstrandofitstargetDNAandtheactionofanexonuclease-deficientDNApolymerasetoextendthe3′endatthenickanddisplacethedownstreamDNAstrand(Walkeretal,1992).Transcription-mediatedamplification(TMA)usesanRNApolymerasetomakeRNAfromapromoterengineeredintheprimerregion,areversetranscriptasetoproducecomplementaryDNAfromtheRNAtemplatesandRNaseHtoremovetheRNAfromcDNA(Guatellietal,1990).Intherollingcircleamplification(RCA),aDNApolymeraseextendsaprimeronacirculartemplate,generatingtandemlylinkedcopiesofthecomplementarysequenceofthetemplate(Fire&
Xu,1995).However,theseisothermalnucleicacidamplificationmethodsalsohavetheirlimitations.Mostofthemhavecomplicatedreactionschemes.Inaddition,theyareincapableofamplifyingDNAtargetsofsufficientlengthtobeusefulformanyresearchanddiagnosticapplications.
Inlivingorganisms,aDNAhelicaseisusedtoseparatetwocomplementaryDNAstrandsduringDNAreplication(Kornberg&
Baker,1992).WehavedevisedanewisothermalDNAamplificationtechnology,helicase-dependentamplification(HDA),bymimickingnature.HDAusesaDNAhelicasetoseparatedoublestrandedDNA(dsDNA)andgeneratesingle-strandedtemplatesforprimerhybridizationandsubsequentextension.AstheDNAhelicaseunwindsdsDNAenzymatically,theinitialheatdenaturationandsubsequentthermocyclingstepsrequiredbyPCRcanallbeomitted.Thus,HDAprovidesasimpleDNAamplificationscheme:
onetemperaturefromthebeginningtotheendofthereaction.Inthisstudy,wepresenttheEscherichiacoliUvrD-basedHDAsystem,whichcanachieveoveramillion-foldamplification.
Results
HDAdesign
ThefundamentalreactionschemeofHDAisshowninFig1
.Inthissystem,strandsofduplexDNAareseparatedbyaDNAhelicaseandcoatedbysinglestrandedDNA(ssDNA)-bindingproteins(SSBs;
Fig1
step1).TwosequencespecificprimershybridizetoeachborderofthetargetDNA(Fig1
step2).DNApolymerasesextendtheprimersannealedtothetemplatestoproduceadsDNA(Fig1
step3).ThetwonewlysynthesizeddsDNAproductsarethenusedassubstratesbyDNAhelicases,enteringthenextroundofthereaction(Fig1
step4).Thus,asimultaneouschainreactionproceedsresultinginexponentialamplificationoftheselectedtargetsequence.
Figure1
SchematicdiagramofHDA.TwocomplementaryDNAstrandsareshownastwolines:
thethickoneisthetopstrandandthethinoneisthebottomstrand.1:
Ahelicase(blacktriangle)separatesthetwocomplementaryDNAstrands,whichareboundbySSB(grey(more...)
E.coliUvrDhelicasewaschosenastheDNAhelicaseforourfirstHDAsystembecauseitcanunwindblunt-endedDNAfragments(Runyon&
Lohman,1989).TheSSBintheHDAreactioniseitherbacteriophageT4gene32protein(Casas-Finet&
Karpel,1993)orRB49gene32protein(Desplatsetal,2002).
AmplificationofatargetsequencefromplasmidDNA
TwoM13/pUC19universalprimers(1224and1233)wereusedinanHDAreactiontoamplifyselectivelya110basepair(bp)targetsequencefromaderivativeofpUC19plasmid.Inafirststep,substrateDNAwasmixedwiththeprimersforheatdenaturationandsubsequentannealing.ThecomponentBmixturecontainingkeyenzymes,suchasE.coliUvrDhelicaseplusitsaccessoryproteinMutL,phageT4gene32proteinandtheexo−KlenowfragmentofDNApolymeraseI,wasthenaddedintocomponentA.Aftera1hrincubationperiodat37°
C,a110-bpamplificationproductwasobservedona2%agarosegel(Fig2
lane1).SequencingresultsconfirmedthatitmatchedthetargetDNAsequence.
Figure2
ElectrophoresisofHDAproductsamplifiedfromplasmidDNA.Atwo-stepHDAreaction,witha1hincubationat37°
C,wasperformedinthepresenceofallcomponents(lane1)includingapUC19-derivedplasmidDNA(0.035pmol),primer-1224(10pmol)(more...)
TodeterminetheessentialelementsintheHDAreaction,eachkeycomponentwasomittedfromthereaction.IntheabsenceofUvrDhelicase,noamplificationwasobserved(Fig2
lane2),confirmingthathelicaseisrequiredfortheamplification.IntheabsenceofaccessoryproteinMutL,noamplificationproductwasobserved(Fig2
lane3),suggestingthatUvrDhelicasemediated-amplificationrequiresMutL.Invivo,MutL,themastercoordinatorofmismatchrepair,recruitsUvrDhelicasetounwindtheDNAstrandcontainingthereplicationerror(Lahueetal,1989).MutLstimulatesUvrDhelicaseactivitymorethantenfoldbyloadingitontotheDNAsubstrate(Mechanicetal,2000).IntheabsenceofT4gene32protein,againnoamplificationproductwasobserved(Fig2A
lane4),indicatingthatSSBisrequiredinthisreaction,probablytopreventreassociationofthecomplementaryssDNAtemplatesat37°
C.IntheabsenceofATP,noamplificationproductwasdetected,indicatingthatthehelicasecofactorisessentialforHDA.Targetsequencesupto400bpcanbeefficientlyamplifiedfromplasmidDNA,beyondwhichtheyielddropsmarkedly(datanotshown).
AmplificationoftargetsequencesfromgenomicDNA
TotestwhetherHDAcanbeusedtoamplifyaspecificsequencefrommorecomplexDNAsamples,suchasbacterialgenomicDNA,theE.coliUvrD-basedHDAsystemwasusedtoamplifya123-bpfragmentfromanoralpathogen,Treponemadenticola.ArestrictionendonucleasegeneencodingahomologueofearIR(GenBankaccessionnumber:
TDE0228)waschosenasthetargetgene.TheamplificationpowerofthecurrentHDAsystemwasalsodeterminedbydecreasingtheamountofT.denticolagenomicDNA.Theamountoftemplatewasvariedfrom107to103copiesoftheT.denticolagenome.Ingeneral,theintensitiesoftheHDAproductdecreasedastheinitialcopynumberwaslowered(Fig3A
).With103copiesofinitialtarget,about10ngofproductsweregenerated,whichcorrespondsto1010moleculesofthe123-bpfragment.Thus,thecurrentHDAsystemdescribedhereiscapableofachievingovertenmillion-foldamplification.Thenegativecontrol,containingnoT.denticolagenomicDNA,showednotraceofamplifiedproducts,provingthespecificityandreliabilityofHDA.
Figure3
ElectrophoresisofHDAproductsamplifiedfrombacterialgenomicDNA.(A)Amplificationofa123-bptargetsequencefromT.denticolagenomicDNA.Atwo-stepHDAreaction,witha3hincubationat37°
C,wasperformedinthepresenceofprimer(more...)
InadditiontoT.denticola,theE.coliUvrD-basedHDAsystemcanamplifytargetsequencesfromvariousgenomicDNAsisolatedfromHelicobacterpylori,E.coli,Neisseriagonorrhoeae,Brugiamalayiandhumancells(datanotshown).
OnetemperatureHDA
AshelicasesareabletounwindduplexDNAenzymatically,wetestedwhethertheentireHDAreactioncouldbecarriedoutatonetemperaturewithoutpriorheatdenaturation.Anotherregion(102bp)oftheearIRhomologuegenewaschosenastarget.ComponentBwasaddedtoAeitherimmediatelyorafteradenaturationstep.Theyieldoftheone-stepHDAamplificationwasabout40–60%ofthetwo-s
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