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1、MultiplexFluorescenceMeltingCurveAnalysisforMutationDetectionwithDual-Labeled,Self-QuenchedProbes112121QiuyingHuang,ZanzanLiu,YiqunLiao,XiaoyunChen,YiZhang,QinggeLi*1EngineeringResearchCenterofMolecularDiagnostics,MinistryofEducation,DepartmentofBiomedicalSciences,Schoolo
2、fLifeSciences,andtheKeyLaboratoryoftheMinistryofEducationforCellBiologyandTumorCellEngineering,XiamenUniversity,Xiamen,Fujian,China,2InstituteforBiomedicalResearch,XiamenUniversity,Xiamen,Fujian,ChinaAbstractProbe-basedfluorescencemeltingcurveanalysis(FMCA)isapowerfultool
3、formutationdetectionbasedonmeltingtemperaturegeneratedbythermaldenaturationoftheprobe-targethybrid.Nevertheless,thecolormultiplexing,probedesign,andcross-platformcompatibilityremaintobelimitedbyusingexistingprobechemistries.Weherebyexploredtwodual-labeled,self-quenchedpro
4、bes,TaqManandshared-stemmolecularbeacons,intheirabilitytoconductFMCA.BothprobescouldbedirectlyusedforFMCAandreadilyintegratedwithclosed-tubeampliconhybridizationunderasymmetricPCRconditions.ImprovedflexibilityofFMCAbyusingtheseprobeswasillustratedinthreerepresentativeappl
5、icationsofFMCA:mutationscanning,mutationidentificationandmutationgenotyping,allofwhichachievedimprovedcolor-multiplexingwitheasyprobedesignandversatileprobecombinationandallwerevalidatedwithalargenumberofrealclinicalsamples.Theuniversalcross-platformcompatibilityofthesepr
6、obes-basedFMCAwasalsodemonstratedbya4-colormutationgenotypingassayperformedonfivedifferentreal-timePCRinstruments.Thedual-labeled,self-quenchedprobesofferedunprecedentedcombinedadvantageofenhancedmultiplexing,improvedflexibilityinprobedesign,andexpandedcross-platformcompa
7、tibility,whichwouldsubstantiallyimproveFMCAinmutationdetectionofvariousapplications.Citation:HuangQ,LiuZ,LiaoY,ChenX,ZhangY,etal.(2011)MultiplexFluorescenceMeltingCurveAnalysisforMutationDetectionwithDual-Labeled,Self-QuenchedProbes.PLoSONE6(4):e19206.doi:10.1371/journal.
8、pone.0019206Editor:IgorMokrousov,St.PetersburgPasteurInstitute,RussianFederationReceivedJanuary2
