核酸恒温扩增技术HDA.docx

核酸恒温扩增技术HDA.docx

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核酸恒温扩增技术HDA

EMBORep.2004August;5（8）:

795–800.

Publishedonline2004July9.doi:

10.1038/sj.embor.7400200.

PMCID:

PMC1249482

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

∙ OtherSections▼

oAbstract

oIntroduction

oResults

oDiscussion

oMethods

oSupplementaryMaterial

oReferences

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.

∙ OtherSections▼

oAbstract

oIntroduction

oResults

oDiscussion

oMethods

oSupplementaryMaterial

oReferences

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