PCR技术及其应用PPT课件

.,PCR技术在医学中的应用,医学分子细胞生物学研究方法,.,WhatisPCR,HowtoPCR,WhydoPCR,.,Developedin1983byKaryMullis,polymerasechainreaction(PCR),1993年的诺贝尔化学奖,.,AnimationofPCR,.,PCRReagents,TemplateDNA,Primers,dNTP,DNAPolymerase,Buffer,Mg2+,.,Questions,Annealling,DNApolymerase,v.s.,WhyprimersareessentialforDNApolymerase?,.,Initializationstep,Denaturationstep,Annealingstep,Elongationstep,Finalelongation,Finalhold,PCRprocedure&stages,2040Cycles,Exponentialamplification,Levelingoffstage,Plateau,.,EvolutionofmachinesforPCR,1,2,3,4,.,PCROptimization,Contamination,Hairpins,Polymeraseerrors,Magnesiumconcentration,Non-specificpriming,Primerdimers,Deoxynucleotides,GC-richTemplate,ProofreadingDNAPolymerasePfu,KOD,DMSO,betaineRedesignPrimers,Hot-StartPolymerase,.,PCROptimization:PrimerDesignSoftwares,Primerpremier:,e-PCR(NCBI),Oligo7:,Primer3:,Commandlinestyledesigntool,Multi-Oligodesignforprobesorprimers,SinglepairedprimersdesignforPCR,Avoidnon-specificamplificationbyblast,.,Primersforamplificationofhumanb-actingenomicDNAregion,.,PrimerDesign:avoidfailureofamplification,5-primer:stem-loopstructurewithhighmeltingtemperature,.,Highdifferencebetweenproductandprimermeltingtemperatures.,3-endForwardPrimerdimer.,PrimerDesign:avoidfailureofamplification,3-enddimerbetweentheForwardandReverseprimers.,TerminalstabilityoftheForwardPrimeristoohigh.,.,PrimerDesign:avoidfailureofamplification,.,PCRmethods,.,IsothermalPCR,ReverseTranscriptionPCR,FrequentlyusedPCRmethods,Real-TimePCR(quantitativePCR),Overlap-extensionPCR,NestedPCR,.,FrequentlyusedPCRmethods,ReverseTranscriptionPCR,.,Overlap-extensionPCR,FrequentlyusedPCRmethods,.,NestedPCR,FrequentlyusedPCRmethods,.,FrequentlyusedPCRmethods,Real-TimePCR(quantitativePCR),Detectfluorescenceforeachcycle,Quantitaivebyamplificationcurve,Quanlitycontrol,.,Real-TimePCR,double-strandedDNA-bindingdye,.,Real-TimePCR(quantitativePCR),Detectfluorescenceforeachcycle,.,Real-TimePCR(quantitativePCR),Quanlitycontrolbymeltingcurve,.,Fluorescentreporterprobe,Real-TimePCR,.,Real-TimePCR,PathogenDiagnosis,microRNADiagnosis,heredopathiaDiagnosis,.,Iso-ThermalPCR,Iso-Thermal,Fast,Sensitive,LAMP/RPA,.,Loop-mediatedisothermalamplification(LAMP),.,Recombinasepolymeraseamplification,Twoprimers,Quantitativable,Sensitive,LongPrimers,.,ApplicationofPCR,.,ApplicationofPCR,SelectiveDNAisolation,AmplificationandQuantification,PCRindiagnosisofdiseases,PCRinforensicscience,.,PCRinforensicscience,variablenumberoftandemrepeats,MitochondriaSequencing,ShortTandemRepeat,.,MitochondriaSequencing,PCRinforensicscience,ShortTandemRepeat,variablenumberoftandemrepeats,.,PCRinforensicscience,Sexdiscernment,Y-chromosomespecificDNA,.,MedicalapplicationofPCR,.,Medicalapplications,Genetictesting,Tissuetyping,Tumortargettyping,.,CellFreeDNAanalysis,.,majorcurrenttechniquesforanalyzingfetalchromosomes,.,PCRindiagnosisoffetalheredopathia,.,Babygenderbloodtests,.,Fetalsexdiscernment,Chorionicvillussampling,Obstetricultrasonography,Cell-freefetalDNA,afterthe13thweekofpregnancyAccuracy:9