1、PCR技术及其应用技术及其应用What is PCRHow to PCRWhy do PCRDeveloped in 1983 by Kary Mullis polymerase chain reaction(PCR)1993年的诺贝尔化学奖Animation of PCRPCR ReagentsTemplate DNAPrimersdNTPDNA PolymeraseBufferMg2+QuestionsAnneallingDNA polymerasev.s.Why primers are essential for DNA polymerase?Initialization stepDen
2、aturation stepAnnealing stepElongation stepFinal elongationFinal holdPCR procedure&stages2040 CyclesExponential amplificationLeveling off stagePlateau Evolution of machines for PCR 1234PCR OptimizationContaminationHairpinsPolymerase errorsMagnesium concentrationNon-specific primingPrimer dimersDeoxy
3、nucleotidesGC-rich TemplateProofreading DNA PolymerasePfu,KOD DMSO,betaineRedesign PrimersHot-Start PolymerasePCR 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 PCRAv
4、oid non-specific amplification by blastPrimers for amplification of human b b-actin genomic DNA regionPrimer Design:avoid failure of amplification5-primer:stem-loop structure with high melting temperatureHigh difference between product and primer melting temperatures.3-end Forward Primer dimer.Prime
5、r Design:avoid failure of amplification3-end dimer between the Forward and Reverse primers.Terminal stability of the Forward Primer is too high.Primer Design:avoid failure of amplificationPCR methodsIsothermal PCRReverse Transcription PCR Frequently used PCR methodsReal-Time PCR(quantitative PCR)Ove
6、rlap-extension PCR Nested PCRFrequently used PCR methodsReverse Transcription PCR Overlap-extension PCR Frequently used PCR methodsNested PCR Frequently used PCR methodsFrequently used PCR methodsReal-Time PCR(quantitative PCR)Detect fluorescence for each cycleQuantitaive by amplification curveQuanl
7、ity controlReal-Time PCRdouble-stranded DNA-binding dyeReal-Time PCR(quantitative PCR)Detect fluorescence for each cycleReal-Time PCR(quantitative PCR)Quanlity control by melting curveFluorescent reporter probeReal-Time PCRReal-Time PCRPathogen DiagnosismicroRNA Diagnosisheredopathia DiagnosisIso-Th
8、ermal PCRIso-ThermalFastSensitiveLAMP/RPALoop-mediated isothermal amplification(LAMP)Recombinase polymerase amplificationTwo primersQuantitativableSensitiveLong PrimersApplication of PCRApplication of PCRSelective DNA isolationAmplification and QuantificationPCR in diagnosis of diseasesPCR in forens
9、ic sciencePCR in forensic sciencevariable number of tandem repeatsMitochondria SequencingShort Tandem RepeatMitochondria SequencingPCR in forensic scienceShort Tandem Repeatvariable number of tandem repeatsPCR in forensic scienceSex discernmentY-chromosome specific DNAMedical application of PCRMedic
10、al applicationsGenetic testingTissue typingTumor target typingCell Free DNA analysismajor current techniques for analyzing fetal chromosomesPCR in diagnosis of fetal heredopathia Baby gender blood testsFetal 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 discernmentProcedureDYS14 geneSRY geneDAZ gene
