PCR技术及其应用.pptx
PCR技术在医学中旳应用医学分子细胞生物学研究措施
WhatisPCRHowtoPCRWhydoPCR
Developedin1983byKaryMullispolymerasechainreaction(PCR)1993年旳诺贝尔化学奖
AnimationofPCR
PCRReagentsTemplateDNAPrimersdNTPDNAPolymeraseBufferMg2+
QuestionsAnneallingDNApolymerasev.s.WhyprimersareessentialforDNApolymerase?
InitializationstepDenaturationstepAnnealingstepElongationstepFinalelongationFinalholdPCRprocedurestages20~40CyclesExponentialamplificationLevelingoffstagePlateau
EvolutionofmachinesforPCR1234
PCROptimizationContaminationHairpinsPolymeraseerrorsMagnesiumconcentrationNon-specificprimingPrimerdimersDeoxynucleotidesGC-richTemplateProofreadingDNAPolymerasePfu,KOD…DMSO,betaineRedesignPrimersHot-StartPolymerase
PCROptimization:PrimerDesignSoftwaresPrimerpremier:e-PCR(NCBI)Oligo7:Primer3:CommandlinestyledesigntoolMulti-OligodesignforprobesorprimersSinglepairedprimersdesignforPCRAvoidnon-specificamplificationbyblast
Primersforamplificationofhumanb-actingenomicDNAregion
PrimerDesign:avoidfailureofamplification5’-primer:stem-loopstructurewithhighmeltingtemperature
Highdifferencebetweenproductandprimermeltingtemperatures.3-endForwardPrimerdimer.PrimerDesign:avoidfailureofamplification3-enddimerbetweentheForwardandReverseprimers.TerminalstabilityoftheForwardPrimeristoohigh.
PrimerDesign:avoidfailureofamplification
PCRmethods
IsothermalPCRReverseTranscriptionPCRFrequentlyusedPCRmethodsReal-TimePCR(quantitativePCR)Overlap-extensionPCRNestedPCR
FrequentlyusedPCRmethodsReverseTranscriptionPCR
Overlap-extensionPCRFrequentlyusedPCRmethods
NestedPCRFrequentlyusedPCRmethods
FrequentlyusedPCRmethodsReal-TimePCR(quantitativePCR)DetectfluorescenceforeachcycleQuantitaivebyamplificationcurveQuanlitycontrol
Real-TimePCRdouble-strandedDNA-bindingdye
Real-TimePCR(quantitativePCR)Detectfluorescenceforeachcycle
Real-TimePCR(quantitativePCR)Q