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Loop-mediatedisothermalamplificationofDNA1EikenChemicalCo.Ltd,1381-3Shimoishigami,Ohtawara,Tochigi324-0036,Japan,2DepartmentofBiochemistryandMolecularBiology,TheUniversityofTokyo,GraduateSchoolofMedicine,Bunkyo-ku,Tokyo113-0033,Japanand3DepartmentofLaboratoryMedicine,OsakaUniversityMedicalSchool,2-2Yamadaoka,Suita,Osaka565-0871,JapanNextSectionAbstractWehavedevelopedanovelmethod,termedloop-mediatedisothermalamplification(LAMP),thatamplifiesDNAwithhighspecificity,efficiencyandrapidityunderisothermalconditions.ThismethodemploysaDNApolymeraseandasetoffourspeciallydesignedprimersthatrecognizeatotalofsixdistinctsequencesonthetargetDNA.AninnerprimercontainingsequencesofthesenseandantisensestrandsofthetargetDNAinitiatesLAMP.ThefollowingstranddisplacementDNAsynthesisprimedbyanouterprimerreleasesasingle-strandedDNA.ThisservesastemplateforDNAsynthesisprimedbythesecondinnerandouterprimersthathybridizetotheotherendofthetarget,whichproducesastem–loopDNAstructure.InsubsequentLAMPcyclingoneinnerprimerhybridizestotheloopontheproductandinitiatesdisplacementDNAsynthesis,yieldingtheoriginalstem–loopDNAandanewstem–loopDNAwithastemtwiceaslong.Thecyclingreactioncontinueswithaccumulationof109copiesoftargetinlessthananhour.Thefinalproductsarestem–loopDNAswithseveralinvertedrepeatsofthetargetandcauliflower-likestructureswithmultipleloopsformedbyannealingbetweenalternatelyinvertedrepeatsofthetargetinthesamestrand.BecauseLAMPrecognizesthetargetbysixdistinctsequencesinitiallyandbyfourdistinctsequencesafterwards,itisexpectedtoamplifythetargetsequencewithhighselectivity.PreviousSectionNextSectionReceivedFebruary1,2000;RevisedApril8,2000;AcceptedApril15,2000.PreviousSectionNextSectionINTRODUCTIONNucleicacidamplificationisoneofthemostvaluabletoolsinvirtuallyalllifesciencefields,includingapplication-orientedfieldssuchasclinicalmedicine,inwhichdiagnosisofinfectiousdiseases,geneticdisordersandgenetictraitsisparticularlybenefitedbythisnewtechnique.InadditiontothewidelyusedPCR-baseddetection(1,2),severalamplificationmethodshavebeeninvented.Theyincludenucleicacidsequence-basedamplification(NASBA)(3),self-sustainedsequencereplication(3SR)(4)andstranddisplacementamplification(SDA)(5,6).Eachoftheseamplificationmethodshasitsowninnovationtore-initiatenewroundsofDNAsynthesis.Forexample,PCRusesheatdenaturationofdouble-strandedDNAproductstopromotethenextroundofDNAsynthesis.3SRandNASBAeliminateheatdenaturationbyusingasetoftranscriptionandreversetranscriptionreactionstoamplifythetargetsequence.Similarly,SDAeliminatestheheatdenaturationstepincyclingDNAsynthesisbyemployingasetofrestrictionenzymedigestionsandstranddisplacementDNAsynthesiswithmodifiednucleotidesassubstrate.Thesemethodscanamplifytargetnucleicacidstoasimilarmagnitude,allwithadetectionlimitoflessthan10copiesandwithinanhourorso,butstillhaveshortcomingstoovercome(7,8).Theyrequireeitheraprecisioninstrumentforamplificationoranelaboratemethodfordetectionoftheamplifiedproductsduetopoorspecificityoftargetsequenceselection.Despitethesimplicityandtheobtainablemagnitudeofamplification,therequirementforahighprecisionthermalcyclerinPCRpreventsthispowerfulmethodfrombeingwidelyused,suchasinprivateclinicsasaroutinediagnostictool.Ontheotherhand,NASBAand3SR,whichdonotusethermalcycling,arecompromisedinspecificity,resultingmainlyfromthenecessitytousearelativelylowtemperatureof40°Cforamplification.SDAlargelyovercomestheseshortcomingsbyusingfourprimersandisothermalconditionsforamplification,butstillhasweakpoints:increasedbackgroundsduetodigestionofirrelevantDNAcontainedinthesampleandthenecessitytousecostlymodifiednucleotidesassubstrate.Althoughtheuseofmultipleprimers,suchasinnestedPCRandSDA,hasimprovedamplificationspecificityforthetargetsequence,residualco-amplificationofirrelevantsequencesstillcausesageneralsetbackinnucleicacidamplification,particularlyfordiagnosticuse.WehaverecentlydevelopedanovelmethodthatcanamplifyafewcopiesofDNAto109inlessthananhourunderisothermalconditionsandwithgreaterspecificity.Wedescribethemechanism,sensitivityandspecificityofthisamplificationmethod,termedloop-mediatedisothermalamplification(LAMP).PreviousSectionNextSectionMATERIALSANDMETHODSDNAoligonucleotidesPrimerBIPforM13mp18(M13BIP)consistedofthecomplementarysequence(24nt)ofB1,aTTTTlinkerandB2(24nt):5′-CGACTCTAGAGGATCCCCGGGTAC-TTTT-TGTTGTGTGGAATTGTGAGCGGAT-3′.PrimerFIPforM13mp18(M13FIP)consistedofF1c(25nt),aTTTTlinkerandthecomplementarysequenceofF2c(22nt):5′-ACAACGTCGTGACTGGGAAAACCCT-TTTT-GTGCGGGCCTCTTCGCTATTAC-3′.PrimersB3andF3forM13mp18(M13B3andM13F3)were5′-ACTTTATGCTTCCGGCTCGTA-3′and5′-GTTGGGAAGGGCGATCG-3′,respectively.TheprobesusedforSouthernblothybridizationwere5′-AAGCTTGGCACTGGCCGTCGT-3′(M13-281)and5′-GTTACCCAACTTAATCGCCTTGCAGCACAT-3′(M13-333).TheprimersusedforamplificationoftheHBsregionofhepatitisvirusB(HBV)DNAwere5′-GATAAAACGCCGCAGACACATCCTTCCAACCTCTTGTCCTCCAA-3′(HBVBIP),5′-CCTGCTGCTATGCCTCATCTTCTTTGACAAACGGGCAACATACCTT-3′(HBVFIP),5′-CAAAATTCGCAGTCCCCAAC-3′(HBVB3)and5′-GGTGGTTGATGTTCCTGGA-3′(HBVF3).Theprimersusedforprostate-s
本文标题:Loop-mediated-isothermal-amplification-of-DNA
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