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PCR技术在医学中的应用医学分子细胞生物学研究方法WhatisPCRHowtoPCRWhydoPCRDevelopedin1983byKaryMullispolymerasechainreaction(PCR)1993年的诺贝尔化学奖AnimationofPCRPCRReagentsTemplateDNAPrimersdNTPDNAPolymeraseBufferMg2+QuestionsAnneallingDNApolymerasev.s.WhyprimersareessentialforDNApolymerase?InitializationstepDenaturationstepAnnealingstepElongationstepFinalelongationFinalholdPCRprocedure&stages20~40CyclesExponentialamplificationLevelingoffstagePlateauEvolutionofmachinesforPCR1234PCROptimizationContaminationHairpinsPolymeraseerrorsMagnesiumconcentrationNon-specificprimingPrimerdimersDeoxynucleotidesGC-richTemplateProofreadingDNAPolymerasePfu,KOD…DMSO,betaineRedesignPrimersHot-StartPolymerasePCROptimization:PrimerDesignSoftwaresPrimerpremier:e-PCR(NCBI)Oligo7:Primer3:CommandlinestyledesigntoolMulti-OligodesignforprobesorprimersSinglepairedprimersdesignforPCRAvoidnon-specificamplificationbyblastPrimersforamplificationofhumanb-actingenomicDNAregionPrimerDesign:avoidfailureofamplification5’-primer:stem-loopstructurewithhighmeltingtemperatureHighdifferencebetweenproductandprimermeltingtemperatures.3'-endForwardPrimerdimer.PrimerDesign:avoidfailureofamplification3'-enddimerbetweentheForwardandReverseprimers.TerminalstabilityoftheForwardPrimeristoohigh.PrimerDesign:avoidfailureofamplificationPCRmethodsIsothermalPCRReverseTranscriptionPCRFrequentlyusedPCRmethodsReal-TimePCR(quantitativePCR)Overlap-extensionPCRNestedPCRFrequentlyusedPCRmethodsReverseTranscriptionPCROverlap-extensionPCRFrequentlyusedPCRmethodsNestedPCRFrequentlyusedPCRmethodsFrequentlyusedPCRmethodsReal-TimePCR(quantitativePCR)DetectfluorescenceforeachcycleQuantitaivebyamplificationcurveQuanlitycontrolReal-TimePCRdouble-strandedDNA-bindingdyeReal-TimePCR(quantitativePCR)DetectfluorescenceforeachcycleReal-TimePCR(quantitativePCR)QuanlitycontrolbymeltingcurveFluorescentreporterprobeReal-TimePCRReal-TimePCRPathogenDiagnosismicroRNADiagnosisheredopathiaDiagnosisIso-ThermalPCRIso-ThermalFastSensitiveLAMP/RPALoop-mediatedisothermalamplification(LAMP)RecombinasepolymeraseamplificationTwoprimersQuantitativableSensitiveLongPrimersApplicationofPCRApplicationofPCRSelectiveDNAisolationAmplificationandQuantificationPCRindiagnosisofdiseasesPCRinforensicsciencePCRinforensicsciencevariablenumberoftandemrepeatsMitochondriaSequencingShortTandemRepeatMitochondriaSequencingPCRinforensicscienceShortTandemRepeatvariablenumberoftandemrepeatsPCRinforensicscienceSexdiscernmentY-chromosomespecificDNAMedicalapplicationofPCRMedicalapplicationsGenetictestingTissuetypingTumortargettypingCellFreeDNAanalysismajorcurrenttechniquesforanalyzingfetalchromosomesPCRindiagnosisoffetalheredopathiaBabygenderbloodtestsFetalsexdiscernmentChorionicvillussamplingObstetricultrasonographyCell-freefetalDNAafterthe13thweekofpregnancyAccuracy:~90%riskofdamagetothefetusafterthe11th~15thweekofpregnancyafterthe7thweekofpregnancyAccuracy:~98%FetalsexdiscernmentProcedureDYS14geneSRYgeneDAZgene