PCR技术及其应用课件

PCR技技术及其及其应用用PCR技术及其应用PCR技术及其应用PCR技术及其应用Wha1What is PCRHow to PCRWhy do PCRWhat is PCRHow to PCRWhy do PC2Developed in 1983 by Kary Mullis polymerase chain reaction(PCR)1993年的诺贝尔化学奖Developed in 1983 by Kary Mull3Animation of PCRAnimation of PCR4PCR ReagentsTemplate DNAPrimersdNTPDNA PolymeraseBufferMg2+PCR ReagentsTemplate DNAPrimer5QuestionsAnneallingDNA polymerasev.s.Why primers are essential for DNA polymerase?QuestionsAnneallingDNA polymer6Initialization stepDenaturation stepAnnealing stepElongation stepFinal elongationFinal holdPCR procedure&stages2040 CyclesExponential amplificationLeveling off stagePlateauInitialization stepDenaturatio7 Evolution of machines for PCR 1234 Evolution of machines for PCR8PCR OptimizationContaminationHairpinsPolymerase errorsMagnesium concentrationNon-specific primingPrimer dimersDeoxynucleotidesGC-rich TemplateProofreading DNA PolymerasePfu,KOD DMSO,betaineRedesign PrimersHot-Start PolymerasePCR OptimizationContaminationH9PCR Optimization:Primer Design SoftwaresPrimer premier:e-PCR(NCBI)Oligo 7:Primer3:Command line style design toolMulti-Oligo design for probes or primersSingle paired primers design for PCRAvoid non-specific amplification by blastPCR Optimization:Primer Desig10Primers for amplification of human b b-actin genomic DNA regionPrimers for amplification of h11Primer Design:avoid failure of amplification5-primer:stem-loop structure with high melting temperaturePrimer Design:avoid failure o12High difference between product and primer melting temperatures.3-end Forward Primer dimer.Primer Design:avoid failure of amplification3-end dimer between the Forward and Reverse primers.Terminal stability of the Forward Primer is too high.High difference between produc13Primer Design:avoid failure of amplificationPrimer Design:avoid failure o14PCR methodsPCR methods15Isothermal PCRReverse Transcription PCR Frequently used PCR methodsReal-Time PCR(quantitative PCR)Overlap-extension PCR Nested PCRIsothermal PCRReverse Transcri16Frequently used PCR methodsReverse Transcription PCR Frequently used PCR methodsRev17Overlap-extension PCR Frequently used PCR methodsOverlap-extension PCR Frequent18Nested PCR Frequently used PCR methodsNested PCR Frequently used PCR19Frequently used PCR methodsReal-Time PCR(quantitative PCR)Detect fluorescence for each cycleQuantitaive by amplification curveQuanlity controlFrequently used PCR methodsRea20Real-Time PCRdouble-stranded DNA-binding dyeReal-Time PCRdouble-stranded D21Real-Time PCR(quantitative PCR)Detect fluorescence for each cycleReal-Time PCR(quantitative PC22Real-Time PCR(quantitative PCR)Quanlity control by melting curveReal-Time PCR(quantitative PC23Fluorescent reporter probeReal-Time PCRFluorescent reporter probeReal24Real-Time PCRPathogen DiagnosismicroRNA Diagnosisheredopathia DiagnosisReal-Time PCRPathogen Diagnosi25Iso-Thermal PCRIso-ThermalFastSensitiveLAMP/RPAIso-Thermal PCRIso-ThermalFast26Loop-mediated isothermal amplification(LAMP)Loop-mediated isothermal ampli27Recombinase polymerase amplificationTwo primersQuantitativableSensitiveLong PrimersRecombinase polymerase amplifi28Application of PCRApplication of PCR29Application of PCRSelective DNA isolationAmplification and QuantificationPCR in diagnosis of diseasesPCR in forensic scienceApplication of PCRSelective DN30PCR in forensic sciencevariable number of tandem repeatsMitochondria SequencingShort Tandem RepeatPCR in forensic sciencevariabl31Mitochondria SequencingPCR in forensic scienceShort Tandem Repeatvariable number of tandem repeatsMitochondria SequencingPCR in 32PCR in forensic scienceSex discernmentY-chromosome specific DNAPCR in forensic scienceSex dis33Medical application of PCRMedical application of PCR34Medical applicationsGenetic testingTissue typingTumor target typingMedical applicationsGenetic te35Cell Free DNA analysisCell Free DNA analysis36major current techniques for analyzing fetal chromosomesmajor current techniques for a37PCR in diagnosis of fetal heredopathia PCR in diagnosis of fetal here38Baby gender blood testsBaby gender blood tests39Fetal sex discernmentChorionic villus samplingObstetric ultrasonographyCell-free fetal DNAafter the 13th week of pregnancyAccuracy:90%risk of damage to the fetusafter the 11th15th week of pregnancyafter the 7th week of pregnancyAccuracy:98%Fetal sex discernmentChorionic40Fetal sex discernmentFetal sex discernment41ProcedureDYS14 geneSRY geneDAZ geneProcedureDYS14 geneSRY geneDAZ42谢谢谢谢43