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1、PCRPCR技术在医学中的应用技术在医学中的应用医学分子细胞生物学研究方法医学分子细胞生物学研究方法1专业课What is PCRHow to PCRWhy do PCR2专业课Developed in 1983 by Kary Mullis polymerase chain reaction (PCR) 1993年的诺贝尔化学奖3专业课Animation of PCR4专业课PCR ReagentsTemplate DNAPrimersdNTPDNA PolymeraseBufferMg2+5专业课QuestionsAnneallingDNA polymerasev.s.Why primer
2、s are essential for DNA polymerase?6专业课Initialization stepDenaturation stepAnnealing stepElongation stepFinal elongationFinal holdPCR procedure & stages2040 CyclesExponential amplificationLeveling off stagePlateau7专业课 Evolution of machines for PCR 12348专业课PCR OptimizationContaminationHairpinsPolymer
3、ase errorsMagnesium concentrationNon-specific primingPrimer dimersDeoxynucleotidesGC-rich TemplateProofreading DNA PolymerasePfu, KOD DMSO, betaineRedesign PrimersHot-Start Polymerase9专业课PCR Optimization: Primer Design SoftwaresPrimer premier:e-PCR (NCBI)Oligo 7:Primer3:Command line style design too
4、lMulti-Oligo design for probes or primersSingle paired primers design for PCRAvoid non-specific amplification by blast10专业课Primers for amplification of human b b-actin genomic DNA region11专业课Primer Design: avoid failure of amplification5-primer: stem-loop structure with high melting temperature12专业课
5、High 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.13专业课Primer Design: avoid failure of amplification14专业课PCR
6、methods15专业课Isothermal PCRReverse Transcription PCR Frequently used PCR methodsReal-Time PCR (quantitative PCR)Overlap-extension PCR Nested PCR16专业课Frequently used PCR methodsReverse Transcription PCR 17专业课Overlap-extension PCR Frequently used PCR methods18专业课Nested PCR Frequently used PCR methods19
7、专业课Frequently used PCR methodsReal-Time PCR (quantitative PCR)Detect fluorescence for each cycleQuantitaive by amplification curveQuanlity control20专业课Real-Time PCRdouble-stranded DNA-binding dye21专业课Real-Time PCR (quantitative PCR)Detect fluorescence for each cycle22专业课Real-Time PCR (quantitative P
8、CR)Quanlity control by melting curve23专业课Fluorescent reporter probeReal-Time PCR24专业课Real-Time PCRPathogen DiagnosismicroRNA Diagnosisheredopathia Diagnosis25专业课Iso-Thermal PCRIso-ThermalFastSensitiveLAMP / RPA26专业课Loop-mediated isothermal amplification (LAMP)27专业课Recombinase polymerase amplificatio
9、nTwo primersQuantitativableSensitiveLong Primers28专业课Application of PCR29专业课Application of PCRSelective DNA isolationAmplification and QuantificationPCR in diagnosis of diseasesPCR in forensic science30专业课PCR in forensic sciencevariable number of tandem repeatsMitochondria SequencingShort Tandem Rep
10、eat31专业课Mitochondria SequencingPCR in forensic scienceShort Tandem Repeatvariable number of tandem repeats32专业课PCR in forensic scienceSex discernmentY-chromosome specific DNA33专业课Medical application of PCR34专业课Medical applicationsGenetic testingTissue typingTumor target typing35专业课Cell Free DNA anal
11、ysis36专业课major current techniques for analyzing fetal chromosomes37专业课PCR in diagnosis of fetal heredopathia 38专业课Baby gender blood tests39专业课Fetal 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%40专业课Fetal sex discernment41专业课ProcedureDYS14 geneSRY geneDAZ gene42专业课
