1定时定量PCR技术原理概要.pdf

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1、PCRPCRPCRPCR––20VanGuilder,H.D.etal.2008.‚Twenty-fiveyearsofquantitativePCRforgeneexpressionanalysis.‛Biotechniques445:619-26PCRDNAPCRPCRDNAPCRDNADNAPCRDNAPCRPCRDNAPCRPCRPCRDNADNA1PCRPCRDNA––PCRDNAPCRPCRPCRS1PCR1SPCRPCRPCR22PCRPCRPCR2PCR2PCRPCRCp1012PCR101

2、0PCRPCR90%DNA1.81.80.80.8[1+]n‘1’2.0100%1.995%PCRPCRdNTPsPCRPCRPCR3Y-X-3PCR33PCRE=10-1/210-3.32PCRE=10-1/=CpPCR1:101:100DNA2Cp1PCR210Cp~3.3n=3.3444CpCp5CpPCRCp5CpPCRPCRDNA–DNA105PCRPCRDNASYBRGreenIDNAPCR-SYBRGreenIHybProbeSYBRGreenISYBRGreenIDNASYBRGreen

3、IdsDNAPCRdsDNASYBRGreenI666SYBRGreenIPCRSYBRGreenIDNAdsDNASYBRGreenIDNAdsDNAABSYBRGreenIdsDNASYBRGreenICDNASYBRGreenIPCRSYBRGreenISYBRGreenISYBRGreenIdsDNA-ResoLightPCRdsDNA767HybProbe–PCRFRETHybProbeHybProbeFRETPCRHybProbeDNA~470nm1-5HybProbeHybProbe8PCRD

4、NAHybProbe88:HybProbe7TaqMan®PCR3´DNA5´FRETPCR5´999:SimpleProbeHybProbeSimpleProbeSimpleProbeScorpions®PCR--PCRblockerPCRTaqDNAScorpions®-Scorpions®PCRScorpions®PCRScorpionPCRScorpions®PCR/8PCRPCRPCR–/ml[GOI]PCRPCRCpPCRPCRPCRCpCpPCRPCRCpCpCpDNA/RNAmRNAGM

5、OMRSAVRE1010:9PCRDNADNADNAPCRPCRNAPCR1111//10PCRPCR/PCRPCRCp-CallingNA1212:PCRPCRNAPCRDNARNANAMS2RNA10ng/μl11NTC=PCR/PCRsRT-PCRRT-RNADNAPCRPCRMasterPCRMasterMasterPCRRNAcDNAcDNA810RNAPCR123´3´3´3´GCGC40%70%G/CA/T16-24HPLCPCR55-60°C500nM100

6、0bp<300bpPCR<150bpPrimer3http://frodo.wi.mit.edu/primer3/input.htmBLASThttp://www.ncbi.nlm.nih.gov/BLASTPCRPCR2PCRPCRPCRPCRPCRPCRPCRs/13GCPCRPCRPCRPCRCpCpCpCpPCRPCR<10014PCRPCR96384PCR151213:96CpabPCR20μl100442bp0.5-CP96CPPCR50PCR15PCRROXXenonPCR

7、2PCR16RESOLIGHTSYBRMolecularProbes8008203361/40082033618008200577asc.support@roche.comBeijingBranchOfficeGuangzhouBranchOfficeShenyangRepresentativeOffice140383B1460925TowerC2-609,TheTowers,Oriental25F,GIETower,No.403Huanshi14F,HaiyueCityPlazaTowerB,N

8、o.83Plaza,No.1EastChangAnAve.Road(E)ZhongshanRoadBeijing,100738,P.R.ChinaGuangzhou,510095,P.R.ChinaShenyang,110001,P.R.ChinaTel.+861085154100Tel.+862087132600Tel:+862423342133Fax+861085154188Fax+862087132700F