利用寡核苷酸芯片对RoundupReady转基因大豆检测及鉴定技术的研究

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1、中国农业大学博士学位论文利用寡核苷酸芯片对RoundupReady转基因大豆检测及鉴定技术的研究姓名：许小丹申请学位级别：博士专业：食品科学指导教师：罗云波20040601ABSTRACTWithmoreandmorecomplicatedGMOplantsunderresearchandbeingapproved，thetraditionalandwidelyusedPCRdetectionmethodcannolongermeetthedemandstofastdetectagreatquantityofanknownGMO．ThedevelopmentofDNAchips(

2、microarray)makesavailableapowerfulandhighthrou≤；hputdetectionandscreeningmethod，whichisthousandsandhundredstimesmoreefficientthanthetraditionalone．However，theresearchofapplyingmicroarraytechnologytoGMOdetectionisstillatthepnmarystage．Inthepresentstudy，theRoundupReadysoybean，oneofthemodelGMOap

3、provedandwimcleartransgenicinformation，waschosenasmainresearchmaterialand，Btl76andBtllmaizeassupplementaryresearchmaterials，theoligonucleotidemieroarraywasusedintheGMOdetectionandidentificationresearch，andthefollowingkeypointsandproblemswerestudiedanddiscussed．I．15genesincludingscreeninggene，

4、specificgene，speciesspecificgene，constructspecificgene，eventspecificgeneforRoundupReadysoybeangeneboxandotherGMOcontrastmaterialwerechosen，everytargetfragmentwasamplifiedunderGMOtemplateDNAandnon-GMOtemplateDNA．TheresultsshowedthattheDNAqualityextractedfromtheCTABmethodsaccordedwiththedemands

5、ofPCRamplification，andeverytargetamplificationwasthesanleasexpectedwhetheritwasGMOtemplateDNAornon，GMOtemplateDNA．ItCallbeconcludedthatthePCRsystemworkedsmoothlyandthetemplateDNAwasnotcontaminated．Thedirectsequencingresultsdidnotdifferfromthedesignedtargetamplificationsequence．II．Anoligonucle

6、otidemicroarraywasmadetodetectandidentifyRoundupReadysoybcan．Itwasshownthatthesequencingresultsincludedprobesequenceorcomplementarysequence，andthevalidsignalproducedwasthesarueasexpectedwithnodifferencefromtheelectmphoresisresults．Thespecificityofoligonucleotidemicroarraywasgood．Thesensitivit

7、yandthespecificityofoligonucleotidemicroarrayarebetterthanthatoftheelectrophoresismethod．111．Thetwocommonlyusedfluorescencelabeledmethods：therandomprimerlabelingandthePCRreactionlabeling，werecomparedandstudied．Judgedfromspecificity，signalinte