Multiplex digital PCR, breaking the one target per color barrier of quantitative PCR

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2、Larson*Received13thFebruary2011,Accepted20thApril2011DOI:10.1039/c1lc20126cQuantitativepolymerasechainreactions(qPCR)basedonreal-timePCRconstituteapowerfulandsensitivemethodfortheanalysisofnucleicacids.However,inqPCR,theabilitytomultiplextargetsusingdifferentlycoloredfluorescentpr

3、obesistypicallylimitedto4-foldbythespectraloverlapofthefluorophores.Furthermore,multiplexingqPCRassaysrequiresexpensiveinstrumentationandmostoftenlengthyassaydevelopmentcycles.DigitalPCR(dPCR),whichisbasedontheamplificationofsingletargetDNAmoleculesinmanyseparatereactions,isanattra

4、ctivealternativetoqPCR.HerewereportanovelandeasymethodformultiplexingdPCRinpicolitredropletswithinemulsionsgeneratedandreadoutinmicrofluidicdevicesthattakesadvantageofboththeveryhighnumbersofreactionspossiblewithinemulsions(>106)aswellasthehighlikelihoodthattheamplificationofonlyas

5、ingletargetDNAmoleculewillinitiatewithineachdroplet.Byvaryingtheconcentrationofdifferentfluorogenicprobesofthesamecolor,itispossibletoidentifythedifferentprobesonthebasisoffluorescenceintensity.Addingmultiplecolorsincreasesthenumberofpossiblereactionsgeometrically,ratherthanlinearl

6、yaswithqPCR.Accurateandprecisecopynumbersofuptosixteenpercellweremeasuredusingamodelsystem.A5-plexassayforspinalmuscularatrophywasdemonstratedwithjusttwofluorophorestosimultaneouslymeasurethecopynumberoftwogenes(SMN1andSMN2)andtogenotypeasinglenucleotidepolymorphism(c.815A>G,SMN1)

7、.ResultsofapilotstudywithSMApatientsarepresented.DownloadedbyUnileverR&DShanghaion13October2011Publishedon17May2011onhttp://pubs.rsc.org

8、doi:10.1039/C1LC20126CIntroductiongrowsinmedicallyrelatedfieldssuchasdiagnostics,theabilitytoperformandquantifymultipleamplificationssimultaneous

9、lyTheadventofPCRandreal-timePCRmethodolo