核酸检测技术.ppt

第2章核酸检测技术,核酸检测技术直接对动植物有害生物基因序列和结构进行测定。核酸的分离纯化PCR技术核酸杂交技术,第1节核酸的分离纯化,核酸的分离纯化的原则,保持核酸一级结构的完整性提高核酸制品的纯度,技术路线的设计,破碎细胞的方法,机械（匀浆、超声破、研磨等）,非机械（化学处理、生化法）,沉淀是浓缩核酸最常用且高效的方法。,核酸浓度检测与完整性测定方法,浓度测定紫外分光光度法260nm荧光光度法EB荧光,完整性测定琼脂糖凝胶电泳法：以EB为示踪剂的核酸凝胶电泳结果为依据。基因组DNA片段如发生降解，电泳图呈拖尾状；完整的或降解很少的总RNA电泳图谱中，三条带的荧光强度积分应呈特定的比值,问题一：DNA样品不纯抑制后续酶解和PCR反应,DNA提取常见问题,DNA提取常见问题,问题二：DNA降解,DNA提取常见问题,问题三：DNA提取量少,真菌,昆虫,第2节PCR扩增技术,PCR,aDNAphotocopier,PCR技术简史,DNA的复制核酸体外扩增的设想聚合酶链反应的发明,1985年，美国PE-Cetus公司的Mullis等人发明了聚合酶链反应（PCR）最初采用E-coliDNA聚合酶进行PCR，由于该酶不耐热，使这一过程耗时，费力，且易出错耐热DNA聚合酶的应用使得PCR能高效率的进行，随后PE-Cetus公司推出了第一台PCR自动化热循环仪1993年，Mullis等因此项技术获诺贝尔化学奖,PCR技术简史,DNA的复制核酸体外扩增的设想聚合酶链反应的发明,KaryB.Mullis（1944）,1989年美国Science杂志列PCR为十余项重大科学发明之首,比喻1989年为PCR爆炸年,Mullis荣获1993年度诺贝尔化学奖。,1.PCR的基础,Polymerase,Chain,Reaction,PCR,TheonlyenzymeusedinthisreactionisDNApolymerase.,Polymerase,Chain,Reaction,Theproductsofthefirstreactionbecomesubstratesofthefollowingone,andsoon.,PCR,Polymerase,Chain,Reaction,PCR,Components：ThermostableDNAPolymerase，TargetDNA，PairofPrimers，dNTPs，Mg+ions，Buffersolution,Denaturation：ThetargetDNA(template)isseparatedintotwostandsbyheatingto95Primerannealing:Thetemperatureisreducedtoaround55toallowtheprimerstoanneal.Polymerization(elongation,extension):Thetemperatureisincreasedto72foroptimalpolymerizationstepwhichusesupdNTPsandrequiredMg2+.,ThePCRcycle:ThreedifferentstepsineachPCRcycle,PCR的扩增？,HowdoesPCRwork,HowdoesPCRwork,Thethirdcycle,Thefourthcycle,2.PCR的类型,多重PCR（multiplexPCR）巢式PCR（nestedPCR）定量PCR（quantitativePCR）不对称PCR（asymmetricPCR）反向PCR（inversePCR）逆转录PCR（reversetranscriptionPCR）OverlapPCR,PCRusingseveralprimerpairsSIMULTANEOUSLYTypicallygeneratesaproductbandforeachprimerpair,多重PCR（multiplexPCR）:What,2.1多重PCR（multiplexPCR）,MultiplexPCR:Why,Detectseveralgenesatonceeg.transgenicplantscreenInternalcontrolsVERYimportantTellsyouhowwellthePCRreactionworkedReduces“falsenegatives”Reduces“falsepositives”,多重PCR（multiplexPCR）,MultiplexPCR:How,SameasregularPCRCareinprimerdesignMuchgreaterchanceofprimer-dimersMuchgreaterchanceofartifactsAnnealingtemperaturesmustbeclose,多重PCR（multiplexPCR）,ATypicalMultiplexPCRReaction,SterileWater34.0ul10XPCRBuffer5.0ulMgCl2(50mM)2.5uldNTPs(10mMeach)1.0ulPrimer1FWD1.0ulPrimer1REV1.0ulPrimer2FWD1.0ulPrimer2REV1.0ulPrimer3FWD1.0ulPrimer3REV1.0ulDNAPolymerase0.5ulDNATemplate1.0ulTotalVolume50.0ul,多重PCR（multiplexPCR）,MultiplexPCR:Example,ThreeprimerpairsControl,resistance,andtraitgenesControlgenefragmentislargestand(almost)faintestTraitgeneissmallestandbrightest,ResistanceGene,TraitGene,ControlGene,Primers,多重PCR（multiplexPCR）,MultiplexPCR:Example,Threeprimerpairs,ResistanceGene,TraitGene,Whicharetransgenic?,ControlGene,Primers,多重PCR（multiplexPCR）,NestedMultiplexPCR,2ndStagePCR,Dilute100foldBetween1stdependentonmutationwithinrecognitionsiteofrestrictionenzymeFormerusedwithsouthernblotexperimentsEvenasmanyrestrictionenzymesareknown,somemutationsitesdonotcorrespondtoany,-Rareendonucleasesaredifficulttoworkwith,andoftenofapoorquality,Restrictionfragmentlengthpolymorphism(RFLP),Restrictionfragmentlengthpolymorphism(RFLP),Modifiedmethod:DiagnosticrestrictionsiteintroducedartificiallybypurposedlyismatchedPCRprimer,140,PCRbasedmethods,RandomAmplifiedPolymorphicDNA(RAPD),RAPDisoftenusedtoshowrelatednessamongDNApopulations.Inthisprocedurearbitrary(random)primersareusedduringPCRtoproduceafingerprintoftheDNA.Asingleprimerisusedwhichmustannealin2placesontheDNAtemplateandregionbetweentheprimerswillbeamplified.,Theprimers(8-10nt)arelikelytoannealinmanyplacesonthetemplateDNAandwillproduceavarietyofsizesofamplifiedproducts.Amplifiedproductsareseparatedbyagarosegelelectrophoresisandvisualized.Ifthesampleshavesimilargeneticmakeupthenthepatternofbandsonthegelwillbesimilarandviceversa.Thi