DNA重组技术的研究综述-张亚旭

20122157～63CurrentBiotechnologyISSN2095-櫅櫅櫅櫅櫅櫅櫅櫅櫅櫅櫅櫅櫅櫅櫅櫅櫅櫅櫅櫅櫅櫅櫅櫅殯殯殯殯2341TechniquesandMethods2011-11-292012-01-088632006AA020903。。[e-mail]510663DNADNA。DNA－、、、、。DNADOI10．3969/j．issn．2095-2341．2012．01．11ResearchSummaryonDNARecombinationTechnologyZHANGYa-xuGuangzhouFulenGenCo．Ltd．Guangzhou510663ChinaAbstractTheresearchandapplicationofDNArecombinationtechnologyi．e．DNAcloningtechnologyisanimportantbranchofthedevelopmentofmodernbiologyandoutstandingareaofmolecularbiology．ThepaperstartedfromthetypesandtherelatedbiologicalconceptsofDNArecombinationsummarizedthetraditionaldigestion-ligationclassicalcloningmethodsite-specificrecombinationcloningmethodandhomologousrecombinationcloningmethodhadbeenreportedatpresent．Theaspectsofrespectiveprinciplesstepscharacteristicsandpracticalapplicationswerefocusedon．Finallyaninductionofrespectivemeritsanddrawbacksandapplicationscopesofthesemethodsandthereviewforachievementsandtheprospectforfutureweredescribed．KeywordsDNArecombinationgenecloningclassicalcloningsite-specificrecombinationcloninghomologousrecombinationcloningDNA。DNAinvivo。DNAinvitro。DNA1。geneDNA、、DNA。DNA。DNA。geneengineeringbiotechnologyDNA1。DNA、、、。DNA、。、DNA、、。DNA。DNA。DNADNA、、。12070DNADNArestrictionendonucleaseDNAstickyendbluntendDNADNADNADNADNADNAclone2。－。PCR、DNA。、、、3β2m4Gt1。DNA。DNA。22．12．1．1univectorplas-mid-fusionsystemUPSLiu51998———UPSDNA。———pUNIpHOST。－6。、DNADNA。PlCre-loxP。Cre34bploxP5'-ATAACT-TCGTATAGCATACATTATACGAAGTTAT-3'Pl。Cre、Cre-loxP。UPSDNA56。78GFP、。2．1．2GatewaycloningsystemGatewayInvitrogen1999910λBPLRattB×attP←→attL×attR。4attGatewayλ11。BP85CurrentBiotechnologyλintegraseIntintegrationhostfactorIHFBPattBDNAattPdonorvectorattL1--attL2。ccdB。LRInt、IHFλXisLRattLattRdestinationvectorccdBattB1--attB2。attP。LRGatewayDNA、。DNAcDNA、DNAPCRattBPCR。Gateway、、、。Gateway。1213～15。。11。。。。2．1．3Gateway2004GeneCopoeiaRec-JoinGateway1617GatewayattRattR“－－”。attDNAPCRatt。DNABPRecJoinRecJoin。－ORFORF。18。2．2、1920。。。2．2．1mating-as-sistedgeneticallyintegratedcloningMAGICLiElledge212005DNA———MAG-IC。DNAⅠ———SceⅠDNA。LiElledge95DNAPCRMAGIC。、MAGIC21。MAGICDNADNA。。MAGIC21。。Paddison22RNAshorthairpinRNAshRNA。2．2．2se-quenceandligation-independentcloningSLICLiElledge232007SLIC。RecAinvitroho-mologousrecombinationRecAsingle-strandannealingSSADNA。T4DNA5'→3'DNADNA。SILC510DNA23。SILCDNARecADNA。242008SLICpUC18LacZSLICDNA。25SLICgsiB。2．2．3isothermalsingle-re-actionmethodGibson262009DNA———。50℃T55'DNAPhusionDNATaqDNADNADNA26。DNA40bpDNA。2．2．4In-FusionCloneEZClontechIn-FusionPCRCloningKitGen-ScriptCloneEZPCRCloningKitDNA1527～29。In-FusionPCRIn-FusionDNA3'→5'3'PCRSSA。。、PCR15DpnⅠPCR2∶1In-FusionTE。5～20ngDNA。In-FusionPCRDNA。、30。06CurrentBiotechnology、、、31～34。。2．2．5DNAco-transformationOlieric352010。13～20PCR2．5∶1。NC。6、835。GatewayLIC。DNA。3。DNADNA1。1DNATable1ComparisonofdifferentDNArecombinationtechnology．DNAUPS、、Gateway、、、、RecJoin、、、MAGICDNA、SLICT4DNADNAIsothermalsingle-reactionDNA40bpIn-FusionCloneEZDNA15DNA13～2016DNA、、、、。DNA。4036～38。1985———HumanGenomeProjectHGPDNA。DNA、。。DNADNADNA。1．M．200079－80．2．M．2002215－217．3．β2mJ．2001174384－385．4．Gt1J．2002281110－114．5LiuQLiMZLeibhamDetal．．Theunivectorplasmid-fusionsystemamethodforrapidconstructionofrecombinantDNAwithoutrestrictionenzymesJ．Curr．Biol．199881300－1309．6．———DNAJ．2002225481－484．7．GFPJ．2003113－16．8．UPSJ．20053184－7．9HartleyJLBraschMA．Recombinationalcloningusingengi-neeredrecombinationsitesP．US005888732A1999－03－30．10HartleyJLBraschMA．Recombinationcloningusingengi-neeredrecombinationsitesP．US006143557A2000－11－07．11．DNAJ．200323724－27．12．GatewayJ．200423358－364．13．DREB1AGatewayJ．200782205－208．14．GatewaycpJ．2007152356－357．15．GatewayRNAiJ．20112704144－147．16YangSW．Methodsandnucleicacidvectorsforrapidexpres-sionandscreeningofcDNAclonesP．US20040115812A12004－06－17．17YangSW．MethodandcompositionsforrapidlymodifyingclonesP．US20060166334A12006－07－27．18．J．2009255701－707．19．J．2000172137－140．20．Gap-RepaipBR322-RedJ．2005325533－537．21LiMZElledgeSJ．MAGICAninvivogeneticmethodfortherapidconstructionofrecombinantDNAmoleculesJ．Nat．Genet．2005373311－319．22PaddisonPJSilvaJMConklinDSetal．．Aresourceforlarge-scaleRNAibasedscreensinmammalsJ．Nature200442825427－431．23LiMZElledgeSJ．HarnessinghomologousrecombinationinvitrotogeneraterecombinantDNAviaSLICJ．Nat．Method200743251－256．24．SLICJ．20084533－36．25．gsiBJ．26CurrentBiotechnology2010325505－511．26GibsonDGYoungLChuangRYetal．．EnzymaticassemblyofDNAmoleculesuptoseveralhundredkilobasesJ．Nat．Method200965343－345．27EvansDHWillerDOYanXD．DNAjoiningmethodP．US7575860B22009－08－18．28LiuWQYangPWangTetal．．Homologousrecombina-tion-basedDNAcloningmethodsandcompositionsP．US20100062495A12010－03－11．29SleightSCBartleyBALieviantJAetal．．In-FusionBioBrickassemblyandre-engineeringJ．Nucl．AcidsResearch20103882624－2636．30．J．2008291100－103．31AuKBerrowNSBlagovaEetal．．Applicationofhigh-throughputtechnologiestoastructuralproteomics-typeanalysisofBacillusanthracisJ．ActaCryst．2006D621267－1275．32ShiraiaMImanaka-YoshidabKSchneiderMDetal．．T-box2amediatorofBmp-Smadsignalinginducedhyaluronansynthase2andTgfβ2expressionan