PCR技術(shù)在醫(yī)學(xué)中的應(yīng)用-課件

1、PCR技術(shù)在醫(yī)學(xué)中的應(yīng)用PCR技術(shù)在醫(yī)學(xué)中的應(yīng)用What is PCRHow to PCRWhy do PCRWhat is PCRHow to PCRWhy do PCDeveloped in 1983 by Kary Mullis polymerase chain reaction (PCR) 1993年的諾貝爾化學(xué)獎(jiǎng)Developed in 1983 by Kary MullAnimation of PCRAnimation of PCRPCR ReagentsTemplate DNAPrimersdNTPDNA PolymeraseBufferMg2+PCR ReagentsTempl

2、ate DNAPrimerQuestionsAnneallingDNA polymerasev.s.Why primers are essential for DNA polymerase?QuestionsAnneallingDNA polymerInitialization stepDenaturation stepAnnealing stepElongation stepFinal elongationFinal holdPCR procedure & stages2040 CyclesExponential amplificationLeveling off stagePlateauI

3、nitialization stepDenaturatio Evolution of machines for PCR 1234 Evolution of machines for PCRPCR OptimizationContaminationHairpinsPolymerase errorsMagnesium concentrationNon-specific primingPrimer dimersDeoxynucleotidesGC-rich TemplateProofreading DNA PolymerasePfu, KOD DMSO, betaineRedesign Primer

4、sHot-Start PolymerasePCR OptimizationContaminationHPCR Optimization: Primer Design SoftwaresPrimer premier:e-PCR (NCBI)Oligo 7:Primer3:mand line style design toolMulti-Oligo design for probes or primersSingle paired primers design for PCRAvoid non-specific amplification by blastPCR Optimization: Pri

5、mer DesigPrimers for amplification of human b-actin genomic DNA regionPrimers for amplification of hPrimer Design: avoid failure of amplification5-primer: stem-loop structure with high melting temperaturePrimer Design: avoid failure oHigh difference between product and primer melting temperatures.3-

6、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 producPrimer Design: avoid failure of amplificationPrimer Design: avoid failure oPCR methodsPCR metho

7、dsIsothermal PCRReverse Transcription PCR Frequently used PCR methodsReal-Time PCR (quantitative PCR)Overlap-extension PCR Nested PCRIsothermal PCRReverse TranscriFrequently used PCR methodsReverse Transcription PCR Frequently used PCR methodsRevOverlap-extension PCR Frequently used PCR methodsOverl

8、ap-extension PCR FrequentNested PCR Frequently used PCR methodsNested PCR Frequently used PCRFrequently used PCR methodsReal-Time PCR (quantitative PCR)Detect fluorescence for each cycleQuantitaive by amplification curveQuanlity controlFrequently used PCR methodsReaReal-Time PCRdouble-stranded DNA-b

9、inding dyeReal-Time PCRdouble-stranded DReal-Time PCR (quantitative PCR)Detect fluorescence for each cycleReal-Time PCR (quantitative PCReal-Time PCR (quantitative PCR)Quanlity control by melting curveReal-Time PCR (quantitative PCFluorescent reporter probeReal-Time PCRFluorescent reporter probeReal

10、Real-Time PCRPathogen DiagnosismicroRNA Diagnosisheredopathia DiagnosisReal-Time PCRPathogen DiagnosiIso-Thermal PCRIso-ThermalFastSensitiveLAMP / RPAIso-Thermal PCRIso-ThermalFastLoop-mediated isothermal amplification (LAMP)Loop-mediated isothermal ampliRebinase polymerase amplificationTwo primersQ

11、uantitativableSensitiveLong PrimersRebinase polymerase amplificatApplication of PCRApplication of PCRApplication of PCRSelective DNA isolationAmplification and QuantificationPCR in diagnosis of diseasesPCR in forensic scienceApplication of PCRSelective DNPCR in forensic sciencevariable number of tan

12、dem repeatsMitochondria SequencingShort Tandem RepeatPCR in forensic sciencevariablMitochondria SequencingPCR in forensic scienceShort Tandem Repeatvariable number of tandem repeatsMitochondria SequencingPCR in PCR in forensic scienceSex discernmentY-chromosome specific DNAPCR in forensic scienceSex

13、 disMedical application of PCRMedical application of PCRMedical applicationsGenetic testingTissue typingTumor target typingMedical applicationsGenetic teCell Free DNA analysisCell Free DNA analysismajor current techniques for analyzing fetal chromosomesmajor current techniques for aPCR in diagnosis of fetal heredopathia PCR in diagnosis of fetal hereBaby gender blood testsBaby gender blood testsFetal sex discernmentChorionic villus samplingObstetric ultrasonographyCell-free fetal DNAafter the 13th week of pregnancyAccuracy: 90%risk of damage to the fetusafter the 11