MGB探针的详细介绍

ABSTRACTHerewedescribethepropertiesofanovelclassofoligonu-cleotideprobescapableofsensitivehybridization-triggeredfluores-cence.Thesefluorogenicprobes,knowncommerciallyasMGBEclipseprobes,arecharacterizedbyhavingaconjugatedminorgroovebinder(MGB)ligandatthe5′-endandafluorophoreatthe3′-end.Additionally,theyhaveanefficientquenchermoietyatthe5′-endthatisusefulwithawidevarietyoffluorescentdyes.Fluores-cenceofthesingle-strandedMGBEclipseprobeisefficientlyquenchedbytheinteractionoftheterminaldyeandquenchergroupswhennothybridized.Uponhybridizationtoacomplementarytarget,theMGBmoleculefoldsintoduplexandhyper-stabilizesit,allowingtheuseofshorter,morespecificprobesequences.The5′-MGB-quenchergroupalsopreventsnucleasedigestionbyTaqDNApoly-meraseduringPCR.Becauseofthehybridization-triggeredfluores-cenceandtheexcellentspecificityimpartedbytheMGB,these5′-MGBEclipseprobeshavegreatversatilityforreal-timePCRap-plications.Thehighsensitivityandspecificityareillustratedusingsinglenucleotidepolymorphismdetection,viralloaddetermination,andgeneexpressionanalysis.INTRODUCTIONReal-timeDNAtargetdetectionduringamplificationhasproventobearobustandefficientmethodforsensitiveandquantitativesequencedetectionandidentification.Amplifica-tionandhybridization,whencombinedinonestep,canbeper-formedinafullyautomated,high-throughput,closed-tubefor-mat.Methodsusedtodateforthistechniquearebasedprimarilyonusingfluorescenceresonanceenergytransfer(FRET)probesthataredesignedtohybridizetoaninternalre-gionofaPCRproductduringtheannealingstage.Theseprobesareeithercleavedinthereaction(asTaqman®MGBprobes)orundergoaconformationchangeinthepresenceofacomplementaryDNAtarget(asMolecularBeacons)andbothutilizeadisruptionofFRET,aphenomenondescribedbyParkhurstandParkhurst(18)andrecentlyreviewedbyDidenko(6).ForTaqmanMGBprobes,the5′-exonucleaseactivityofTaqDNApolymerasecleavestheprobefromthe5′-end,be-tweenthefluorophoreandquencher,thusreleasingfluores-cence(11).Modifiedbases(9)andminorgroovebinder(10)reportedlyimprovetheperformanceoftheTaqmanMGBprobes.Fluorescencequenchingofabeaconprobeisaresultofanintramolecularstemformationthatbringsthefluo-rophoreandquencherincloseproximity(22).Whenhy-bridizedtothetargetDNAstrand,quencherandfluorophoreareseparatedenoughtoproduceafluorescentsignal.Thebeaconprobesare35–40baseslong,includingtherequiredextra10–12basesforhairpinformation.Becauseoftheneedtobalancethestabilitiesofthestem-loopwiththehybridizedprobe,thedesignofsuchprobescanbecomplicated.Methodsthatusedouble-strand-specificfluorophoressuchasSYBR®Green(20)orlabeledprimers(15)cantakeadvan-tageoflessexpensivereagentsbutdonotprovideassuranceofspecificamplification.Toprovethatsignalincreaseisnotduetofalseprimingorprimer-dimerformation,post-PCRstepssuchasaproductmeltingcurveanalysisarerequired.These-quencespecificityprovidedbyfluorogenicprobessuchasTaqmanMGBorMolecularBeaconsisbeneficialforhigh-throughputapplications.Herewedescribeathirdtypeoffluo-rogenicprobeforDNAmeasurementthatwecallMGBEclipseprobes.Wehavebeenexploringthechemistryandapplicationsofoligonucleotideswithaconjugatedminorgroovebinder(MGB)ligand,whichformhyper-stabilizedduplexeswith940BioTechniquesVol.32,No.4(2002)...aforumformanufacturerstodescribethecurrentandpotentialapplicationsofnewresearchinstrumentsorproducts.PRODUCTAPPLICATIONFOCUSMinorGrooveBinder-ConjugatedDNAProbesforQuantitativeDNADetectionbyHybridization-TriggeredFluorescenceI.A.Afonina,M.W.Reed,E.Lusby,I.G.Shishkina,andY.S.BelousovEpochBiosciences,Bothell,WA,andSyntheticGenetics,SanDiego,CA,USABioTechniques32:940-949(April2002)complementaryDNA(1,2,10,12).Thesequencespecificityofthesenovelagentsprovidesasignificantadvantageforhigh-temperatureapplicationssuchasPCR(10).Wehavealsode-velopedanon-fluorescentquencher(theEclipseDarkQuencher,Q)foruseinourfluorogenicMGBEclipseprobes(13).Whenusedashybridizationprobes,theEclipseDarkQuenchercombinationobviatestheneedforthestemstructure,asinMolecularBeacons,andallowstheuseofmuchshorteroligonucleotides.HerewereportthesynthesisandpropertiesofMGBEclipseoligonucleotideswithanattachedfluorophoreanddescribetheirperformanceashybridization-triggeredflu-orogenicprobesforPCR.TheshortlengthanduniquestructureofMGBEclipseprobesgiveverylowfluorescentbackgroundandexcellentspecificity,andthisperformanceisdemonstratedinthereal-timePCRapplicationsofsinglenucleotidepolymor-phism(SNP)detection,viralloaddetermination,andgeneex-pressionanalysis.MATERIALSANDMETHODSTemplatesHumangenomicDNAsfromthepedigreefamilyno.66wereusedtostudytheRRM1SNP(16,17).Allele1homozy-gousDNA(mother),allele2homozygousDNA,andhet-erozygousDNA(son)werepurchasedfromCoriellInstituteofMedicalResearch.OtherhumangenomicDNAsusedintheSNPassaywerepurchasedfromthesameinstitute,withtheexceptionofthe“p53”and“CYP2D6*4”templates.Genotypingwasdonepreviouslyinourlaboratoryusingre-strictionlengthpolymorphism(14)andTaqmanMGBmeth-ods.PCMV-p53andpCMV-p53mt135plasmidscontainingwild-typeandmutantp53G1017AconstructswerefromBDBiosciencesClontech(PaloAlto,CA,USA).CYP2D6*4wild-typeandmutanthuma