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,1,WhatisPCR,HowtoPCR,WhydoPCR,2,Developedin1983byKaryMullis,polymerasechainreaction(PCR),1993年的诺贝尔化学奖,3,AnimationofPCR,4,PCRReagents,TemplateDNA,Primers,dNTP,DNAPolymerase,Buffer,Mg2+,5,Questions,Annealling,DNApolymerase,v.s.,WhyprimersareessentialforDNApolymerase?,6,Initializationstep,Denaturationstep,Annealingstep,Elongationstep,Finalelongation,Finalhold,PCRprocedure&stages,2040Cycles,Exponentialamplification,Levelingoffstage,Plateau,7,EvolutionofmachinesforPCR,1,2,3,4,8,PCROptimization,Contamination,Hairpins,Polymeraseerrors,Magnesiumconcentration,Non-specificpriming,Primerdimers,Deoxynucleotides,GC-richTemplate,ProofreadingDNAPolymerasePfu,KOD,DMSO,betaineRedesignPrimers,Hot-StartPolymerase,9,PCROptimization:PrimerDesignSoftwares,Primerpremier:,e-PCR(NCBI),Oligo7:,Primer3:,Commandlinestyledesigntool,Multi-Oligodesignforprobesorprimers,SinglepairedprimersdesignforPCR,Avoidnon-specificamplificationbyblast,10,Primersforamplificationofhumanb-actingenomicDNAregion,11,PrimerDesign:avoidfailureofamplification,5-primer:stem-loopstructurewithhighmeltingtemperature,12,Highdifferencebetweenproductandprimermeltingtemperatures.,3-endForwardPrimerdimer.,PrimerDesign:avoidfailureofamplification,3-enddimerbetweentheForwardandReverseprimers.,TerminalstabilityoftheForwardPrimeristoohigh.,13,PrimerDesign:avoidfailureofamplification,14,PCRmethods,15,IsothermalPCR,ReverseTranscriptionPCR,FrequentlyusedPCRmethods,Real-TimePCR(quantitativePCR),Overlap-extensionPCR,NestedPCR,16,FrequentlyusedPCRmethods,ReverseTranscriptionPCR,17,Overlap-extensionPCR,FrequentlyusedPCRmethods,18,NestedPCR,FrequentlyusedPCRmethods,19,FrequentlyusedPCRmethods,Real-TimePCR(quantitativePCR),Detectfluorescenceforeachcycle,Quantitaivebyamplificationcurve,Quanlitycontrol,20,Real-TimePCR,double-strandedDNA-bindingdye,21,Real-TimePCR(quantitativePCR),Detectfluorescenceforeachcycle,22,Real-TimePCR(quantitativePCR),Quanlitycontrolbymeltingcurve,23,Fluorescentreporterprobe,Real-TimePCR,24,Real-TimePCR,PathogenDiagnosis,microRNADiagnosis,heredopathiaDiagnosis,25,Iso-ThermalPCR,Iso-Thermal,Fast,Sensitive,LAMP/RPA,26,Loop-mediatedisothermalamplification(LAMP),27,Recombinasepolymeraseamplification,Twoprimers,Quantitativable,Sensitive,LongPrimers,28,ApplicationofPCR,29,ApplicationofPCR,SelectiveDNAisolation,AmplificationandQuantification,PCRindiagnosisofdiseases,PCRinforensicscience,30,PCRinforensicscience,variablenumberoftandemrepeats,MitochondriaSequencing,ShortTandemRepeat,31,MitochondriaSequencing,PCRinforensicscience,ShortTandemRepeat,variablenumberoftandemrepeats,32,PCRinforensicscience,Sexdiscernment,Y-chromosomespecificDNA,33,MedicalapplicationofPCR,34,Medicalapplications,Genetictesting,Tissuetyping,Tumortargettyping,35,CellFreeDNAanalysis,36,majorcurrenttechniquesforanalyzingfetalchromosomes,37,PCRindiagnosisoffetalheredopathia,38,Babygenderbloodtests,39,Fetalsexdiscernment,Chorionicvillussampling,Obstetricultrasonography,Cell-freefetalDNA,afterthe13thweekofpregnancyAccuracy:90%,r