数字PCR技术进展_林彩琴

2412201212PROGRESSINCHEMISTRYVol．24No．12Dec．20122012520127*No．2090506420890020No．20090101120007＊＊[e-mail]*＊＊310058PCR。PCRPCRCT。Fluidigm、Bio-RadPCR、。PCR。PCRQ503TP212.3A1005-281X201212-2415-09RecentAdvanceinDigitalPCRLinCaiqinYaoBo＊＊DepartmentofChemistryZhejiangUniversityHangzhou310058ChinaAbstractDigitalPCRanewtechniquederivedfromtraditionalPCRandquantitativePCRhasrevealedgreatpotentialingeneticprofilingprenataldiagnosiscancerrelatedallelandmutationdetection．DigitalPCRusuallyinvolvestwostepspartitioningoflimitingdilutionsampleintotenstothousandsofindividualreactionsfollowedbyPCRamplificationandendpointdetectiontoidentifythepresenceorabsenceoftemplatemoleculesineachreaction．DifferentwithrealtimeqPCRwhichgreatlydependsoncyclethresholdCTandrequireshouse-keepinggeneandstandardcurveforrelativeandabsolutequantificationdigitalPCRperformsquantificationthroughdirectcountingofthewellswithpositiveamplificationorcalculatingtheconcentrationbasedonPoissonalgorithm．Itminimizesthemeasurementuncertaintycausedbyvariationofamplificationefficiencyandhashigheraccuracyandreproducibility．InrecentyearsgreateffortshavebeenmadetodevelopnewmethodsfordigitalPCRtoimproveitssensitivityandreduceitscost．MoreandmoretechniqueswerecommercializedsoonafteritwasfreshlypublishedbylargecompaniesincludingBio-RadLifetechnologyandsoon．InthisarticletheprincipleofdigitalPCRanditsquantificationmethoddifferenttypesofthistechniqueanditspracticalapplicationsarereviewedanddiscussed．KeywordsdigitalPCRmicrofluidicchipgeneanalysisContents1Introduction2PrincipleofdigitalPCR3Quantificationmethod4ClassificationofdigitalPCR4.1Microchamber/plate4.2IntegratedfluidiccircuitIFC·2416·244.3DropletbaseddigitalPCR5Applications5.1Geneticinstabilityanalysis5.2Earlydiagnosisofcancer5.3Prenataldiagnosis5.4Others6Conclusionandoutlook1polymerasechainreactionPCR20PCR1。90ABIPCRrealtimePCRqPCRPCR/、。qPCR2PCR———CT。qPCR“”。20VogelsteinPCRdigitalPCRdPCR3DNAPCR。qPCRPCRCT5%4PCR。。FluidigmBio-RadPCR45、6789。PCR、、。2PCRPCRPCRPCR。PCRPCR。qPCRPCR。。Vogelstein3PCR961。DNAPCR。PCR。PCRTaqManPCR。1bPCRPCRasymmetricPCRDNA310SNPdigitalsingle-nucleotidepolymorphismdigitalSNP。TaqMan11PCR5。Vogelstein1213PCR———BEAMingbeadsemulsionamplificationmagnetics1c。BEAMingPCR。。。BEAMing12PCR·2417·TaqMan。。Zhong14PCRSMAc．815A＞GPCR4。1PCRaPCRPCRb3cBEAMing12Fig．1SchematicofdigitalPCR．aTwostepsinvolvedPCRondilutedDNAsamplesandfluorescentanalysis．bPrincipleofmolecularbeaconbyadditionoffluorescentprobesthatdiscriminatebetweenWTandmutantalleles3．cPrincipleofBEAMing12．ReprintswerepermittedbytheNationalAcademyofSciencesUSA3qPCRcyclethresholdCT。PCRNt=N01+ECT1N0NtCTE。logNt=logN01+ECT2CT=－1log1+ElogN0+logNtlog1+EPCRECTNtCTN0。PCRPCR。qPCRPCRPCR10。DNADNA。PCRPoissondistribution。p=λkke－λ3λDNApλkDNA。λmλ=cmc。k=0DNAp=e－λ=e－cmpn－fn=e－cm4nf。lncm=ln1－fn5PCR。PCRhouse-keepinggene915—17。PCR。PCRn。DNAn=1021/1001%。n=10410410.01%。·2418·24PCR。4PCRPCR。PCR/818、511191320PCR。4.1/PCRPCRPCR96/384PCR96/38438。PCRn96/384。96/384PCR5μl。Morrison1825mm×75mm3072300μm2a33nl。PCR3841/6424。。4.2PCR、PCR。2000Unger21multilayersoftlithographyMSLpolydimethylsiloxanePDMS2bIFCintegratedfluidiccircuit。IFCPDMS。2006Ottesen5IFCPCR11766.25nl384。67056PCR。Hansen22MSL106PCR10pl440000/cm2。PCRIFC。Men232mm×2mm82000flPCR。4.3PCRPCRPCRemulsionPCR2425DNAPCR。PCRIFCPCR。Vogelstein121326BEAMingPCRPCR。Lu27BEAMingmRNA。Zhou28BEAMingPCR。DNA。Beer2930DNA、PCRPCR。Hindson203120000—20000001nl96TaqManPCR961000/s。Pekin32DNAKRAS。Shen33SlipChipPCR12802.6nlPCR。SlipChip1nl125nl2001200034。Yang3512PCR·2419·PCR。2PCRa/PCROpenArrayTM18bPCRBioMarkTM5cPCRQX100TM20Fig．2StrategiesofdigitalPCRamicrochamber/platedigitalPCROpenArrayTM18bintegratedfluidiccircuitfordigitalPCRBioMarkTM5cdropletdigitalPCRQX100TM20．ReprintswerepermittedbyOxfordUniversityPressTheAmericanAssociationfortheAdvancementofScienceandAmericanChemicalSocietyrespectively5PCR5.1、36。somaticmutationallelicimbalanceAIlossofheterozygosityLOHcopynumbervariationsCNVs。DNA。VogelsteinPCR。KRASPCRKRAS124%4/1023。SNP18q101318qLOH1818qLOH17。ShihovarianserouscarcinomaBRAF599KRAS121337387、39。Hanlon40multiplemyeloma13q141816LOH89%FISH。Yung4135EGFR19L858RPCR30%。kbMb、CNVs42。CNVsHIV-1、。CNVsmicroarraybasedcomparativegenomichybridizationarray-CGHhighdensityoligonucleotidearrayFISHPCR30%PCR2CTCNVs。Ramakrishnan1619CNVsPCR1RNaseP15%。19408HEBB2CNVs14HEBB2CNVs5。Wang431620EGFRCNVs、19L858REGFR·2420·240.02%—9.26%EGFR。Hindson20MRGPRX1、XCYP2D6CNVsHEBB2CNVs。PCRPCR44。5.2VogelsteinPCR、361045—49。。。。。Shih48APCadenomatouspolyposiscoliLOHAPC。APC50%familialadenomatouspolyposisFAP46。APCLOHAPC2823。Shih47325q、1p、8p、15q18q90%5AI55%5qAI67%5qAI。AI。DNADNA、、。Chang5033012216444DNAAISNPAI。Traverso49BAT2646BAT26181737%。6919100%。Dong45BAT26、TP53KRAS3。Qi51PCRmRNA89100%77%。5.3prenataldiagnosis。、、。0.1%—0.9%。1997Lo52Y。。10%—20%DNA。DNA52—54。2007Lo55384PCR21PLAC4mRNASNPrs8130833digital-RNA-SNP211digitalRCD25%。56digitalRMDdigitalNASSDNA。Fan1112PCR·2421·PCR2110%。5718136h1—2。PCRPCR。5.4DNA、。Shendure58Margulies24PCRDNADNAPCR。PCR10010。/。White59PCRDNA、。PCRSpurgeon60IFC18452304PCRPCRDNA