基因表现连续分析法(SAGE)及其在植物研究之应用农业试验所王强生廖.pdf

该【基因表现连续分析法(SAGE)及其在植物研究之应用农业试验所王强生廖 】是由【小屁孩】上传分享，文档一共【6】页，该文档可以免费在线阅读，需要了解更多关于【基因表现连续分析法(SAGE)及其在植物研究之应用农业试验所王强生廖 】的内容，可以使用淘豆网的站内搜索功能，选择自己适合的文档，以下文字是截取该文章内的部分文字，如需要获得完整电子版，请下载此文档到您的设备，方便您编辑和打印。:..??????????SAGE???????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????(cloningbydifference)???????????????????????????????????????????????????????????????????????????????RNA????RT-PCR??????????????????????????????(subtractivehybridization)???EST?parativeESTanalysis)???????????(profiles)????????????(differentialscreening)????????(differentialdisplay,DD)?????????????????????????????????????????????????????????????????????????????????????????????????????mRNA?????Velculescu??(1995)???????????????(serialanalysisofgeneexpression,SAGE)???????????????????????????????SAGE????????????(post-genomicera)????????????SAGE??????????????NlaIII?????????cDNA3’??????9-11????????DNA(???)??????Linker-Ligations?????????????????PCR???????????????????????????????mRNA??????????(abundance)??????????????????????9??????????(49)?????????????????????????15,000–33,000????????????????????????SAGE??????1:..SAGE????????(???)???TotalRNA???mRNA??1.??phenol/chloroform?(?????)??totalRNA???????SAGE???totalRNA???1mg(??mRNA????5%)????2.???DynalCo.????Dynabeads?Oligo(dT)?25???????????polyARNA??????????5?gpolyARNA?????cDNA????3.???????????firststrandedbuffer?dNTPmixture?biotinylatedoligodT????????????SAGE????primer?5?gpolyARNA?????SAGE???65?????10?????RNA??????????3?????MMLVreversetranscriptase?37??????cDNA????4.???3???cDNA:RNA?RNaseH?DNApolymeraseI?????cDNA?????????cDNA????????????????????????????1.?????cDNA???(CATG)???????(frequentcutenzyme,?NlaIII)(?AE)?????????cDNA?streptavidinbeads????polyA?3'?cDNA?2.????cDNA?????????????LinkerA?LinkerB(?linker????TypeIIS???????)?????????TypeIIS????(?FokI)(TE)???3.?????cDNA?Klenowfragment?????(fillin)????(blunt2:..end)???phenol/chloroform????????????cDNA???????ligation?4.?????cDNA?phenol/chloroform???????????????????????(?NlaIII)??????cDNA???????(ditag)??????26-30???(bases)?5.????????cDNA?????????????????cDNA????????????????(sizefractionation)?????200????????????????????????SAGE???????????????DNA??6.????????????????????????????????????????SAGE?????(???)????SAGE??????????????????????BLASTanalysis????????SAGE????????????????????a.??????????(match)?b.???ESTs(expressedsequencetags)?????mRNA???c.???????????mRNA???SAGE???????????????????(transcriptprofiling)?????????????????????????????p53???????(apoptosis)?????SAGE?????????????????????????????Matsumura?(1999)??SAGE?????????????????SAGE???????????????????????SAGE?????????????????????????????????????????Matsumura?(1999)??????????????????????????(profile)??????????????????RNA??????cDNA???SAGE????????10,122??????5,593?????????????????????5,921????????????????SAGE??????????????????????????????(?????)??100?????80%??????cDNA3:..?EST?????????????????????SAGE??????????????100????????????????????(housekeepinggenes)?????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????SAGE???????????????????????????(anaerobictreatment)??????????????????SAGE??????????????2,094??????????1,496??????????????????2,205???????????????1,551??????????????????????????????(????????????)???????24????????????8???????????????6?????????????????????????????SAGE?????????????(10%)?????????????????(20%)(?????????????)(???)?????????????????????????????????????????????????SAGE??????????????????????????????????????????????????????????????????????SAGE????????????????100????????????????????????????????????????SAGE??????RACE(rapidamplifiedcDNAend)???????9-13?????????????4:..????????????(?M13forward?T7promoter)???????????DNA??????PCR???????????????cDNA????9-13?????????????????????????????????????????????SAGE???????1.??????????????????????????2.???????????????????????3.?????????????????????(????????)???(?????????)????????SAGE??????????????????mRNA(-5μg)????????????????????????????????SAGE???????????????????mRNA??????MicroSAGE?(al.,1999)????SAGE?????????????????????????????PCR??????????(ditag)???????????????MicroSAGE?????????????????????????????SAGE??????????????????????????????????????????????????????????????????????????????????????????????????SAGE???????????????????????????????????????????????????????????????????????????????????,.,vanderPerk-deJong,J.,vanderBerg,.,deKloet,.,andVreugdenhil,::1300-,H.,Nirasawa,S.,andTerauchi,(OryzasativaL.)seedlingsusingserialanalysisofgeneexpression(SAGE).:719-,.,Zhang,L.,Vogelstein,B.,andKinzler,:..:484-,L.,Zhou,W.,Velculescu,.,Kern,.,Hruban,.,Hamilton,.,Volgelstein,Kinzler,:1268-????????????????SAGE??????GenzymeMolecularOncology(GMO)????????????????PCR????SAGE?????????????RAGE(rapidanalysisofgeneexpression)?6