核酸检测技术

.,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.