电子克隆的方法

生物技术通报BIOTECHNOLOGYBULLETIN20101cDNA孙淼1赵茂林2(1,100048;2,100097):基因组计划的进展及表达序列标签数据的迅速扩增使得电子克隆方法孕育而生,为进行基因克隆开辟了一条新的路径介绍了表达序列标签和电子克隆的原理及过程,重点分析电子克隆过程中遇到的问题及解决方法,展望其在新基因功能研究中的作用:表达序列标签电子克隆聚类叠连群StrategiesforinSilicoCloningcDNASequencesBasedonESTSunMiao1ZhaoMaolin2(1CollegeofLifeScience,CapitalNormalUniversity,Beijing100048;2BeijingAgricultureBiotechnologyResearchCenter,BeijingAcademyofAgricultureandForestryScience,Beijing100097)Abstrac:tTheprogressofgenomeprojectandtherapidexpansionofexpressedsequencetags(ESTs),makeinsilicocloningbroughton,whichforushasopenedanewpathtogenecloning.Inthisarticle,anoverviewofEST,theprincipleandmethodofinsilicocloningwerediscussed,focusingonanalysisofproblemsandsolutionsduringinsilicocloningprocess,also,itprospectedtherolesinthestudyofthenewgenefunction.Keywords:ExpressedsequencetagsInsilicocloningClusteringContig:20090914:(30370905,30571135),(5032009):,,,;Emai:[e-mail]:,,,,;Emai:[e-mail],cDNAPCR(differentdisplayPCR,DDRTPCR)(representationdifferenceanalysis,RDA)(suppressionsubtractivehybridization,SSH),ESTscDNA,cDNA,,ESTcDNAEST,[1]1(expressedsequencetags,ESTs)cDNA,53cDNAcDNA300-500bp,EST,1991,Adams[2]cDNAESTsDNARNAi22.1电子克隆的基本原理[3](insilicocloningelectroniccloning)!cDNA∀[4](electroniccDNAlibraryscreening)!∀(virtualcloning),(ESTs生物技术通报BiotechnologyBulletin20101),()ESTs,cDNABoguski[5]1994,1996,,,SR508(SRrp508)[6]TECTB[7],2.2电子克隆的方法步骤2.2.1ESTsESTs,ESTs[8][9][10,11]2.2.2ESTsESTs,ESTs,BLAST,NCBI,NCBI()EST,,E(E)[12]EST,EST(1)1ESTdbEST://=unigeneGenBankTIGRGeneIndices(clustering)[13],EST,,NCBIUNIGENESTACKPACKTIGRGeneIndices,[14]ESTclustering112256ESTs147191ESTs,,,ESTs2.2.3ESTsESTs,(contig),EST[15]EST4095%,,,,CAP3(),CAP,contig,DNASTARSeqMan,;,contig,,5020101:cDNA,DNAMANSequencherTM,,,EST,2.2.4contig,contig,:contigESTcontig,ESTEST,Blastn/Blastx,contig,contig,contig,EST,,EST,,2.2.5cDNA,cDNA,:(1)(ORF):NCBIORFFinder()ORFFinder,BioEditDNAMANSequencherESTmRNA,55UTR(untranslatedregion)[16],Kozak[17]ORF5,[9]:5AUG(GCC)GCCA/GCCAUGG;;55;Northen(2):(2)2PromoterScan://mbs.cbrc.jp/research/db/TFSEARCH.htmlCDD://://(3):,,UniGene,UniGene,UniGeneCluster/,!cDNAsources∀(sequencetaggedsite,STS)PCRUniSTS(=unists)UniGene/RH2.2.6PCR,RTPCR,RACE,cDNA,,51生物技术通报BiotechnologyBulletin201013cDNAcDNA,,EST97%[18],,ESTs,cDNA4,,,,,,,[1],..:,2002,7276.[2]AdamsMD,KelleyJM,CocayneJD,eta.lComplementaryDNAsequencing:expressedsequencetagsandhumangenomeproject.Science,1991,252(5013):16511656.[3]GillRW,SanseauP.Rapidinsilicocloningofgenesusingexpressedsequencetags(ESTs).BiotechnolAnnuRev,2000,5:2544.[4]BanfiS,GuffantiA,BorsaniG.HowtogetthebestofdbEST.TrendsGenet,1998,14(2):8081.[5]BoguskiMS,TolstoshevCM,BassettDEJr.GenediscoveryindbEST.Science,1994,265(5181):19931994.[6],,,,.SR∃SFRS12(SRrp508).,2002,29(5):377383.[7],,,,.TECTB.,2003,30(4):317320.[8],,,,.GTPOstab5B.,2001(3):914.[9],,,,..,2002,16(4):295298.[10],,,,.TaLSD1.,2007,27(11):21472152.[11],,,,.2C/(PP2C).,2008,34(5):899903.[12],.[M].:,2008,70.[13]HideW,MillerR,PtitsynA,KelsoJ,GopallakrihnanC,ChristoffelsA.ESTClusteringTutoria.lISMBinHeidelberg,Germany,1999,6.[14],,,,.EST.,2003,30(2):147153.[15]AttwoodTK,ParrySmithDJ,..:,2002,118.[16]SalamovAA,NishikawaT,SwindellsMB.AssessingproteincodingregionintegrityincDNAsequencingprojects.Bioinformatics,1998,14(5):384390.[17]KozakM.Ananalysisof5noncodingsequencesfrom699vertebratemessengerRNAs.NucleicAcidsRes,1987,15(20):81258148.[18],,.ESTcDNA.,2002,25(2):6769.52