几个提高抑制消减杂交(ssh)文库质量的技术改进

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1、万方数据2011年24卷5期VoL24No．5西南农卫学报Southm∞tChinaJournalofA面culturalSciences，1849文章编号：1001—4829(2011)05—1849—04几个提高抑审IJ消减杂交(SSH)文库质量的技术改进董登峰1’。，李杨瑞“，白云彩1(1．广西大学农学院，广西南宁530004；2．广西作物遗传改良生物技术重点实验室，广西南宁530007)摘要：抑制消减杂交(SSH)高灵敏、易操作、高效富集低表达丰度基因和均一化目的基因片断，广泛用干差异表达基因的分离和eDNA文库构建。在实验操作中常出现低表达mRNA和eDN

2、A丢失、大片断连接效率低等问题，构建的文库质量差，文章通过改进萃取操作提高目的产物(mRNA和eDNA)回收率，回收率增加30％以上；通过采用PvPP、高浓度KAc专一性结合和LICl沉淀等措施除去RNA中的多酚和多糖、进而增加可分离mRNA种类和含量，对应的Uni-EST种类增加近3倍，通过采用胶回收并分段连接保护大片断eDNA等措施使文库中转录本丰富度增加，大片段(500bp以上)比例增大，eDNA平均长度增大，文库质量明显提高。关键词：抑制消减杂交(SSH)；文库构建；技术改进中图分类号：S181文献标识码：AImprovementofSomeTechniqu

3、esforEnhancingQualityofSuppressionSubtractiveHybridizationcDNALibraryDONGDeng-fen91一，LIYang-rui2．，BAIYun-eail(1．CollegeofAgriculture，GuangxiUniversity，GuangxiNanning530004。China；2．Key—LabofCropGeneticImprovementandBiotechnolo-盯，GuangxiNanning530007，China)Abstract：Suppressionsubtracfive

4、hybridizationwaswidelyusedtoisolatedifferentiallyexpressedgenesandconstructeDNAlibrariesatp阱entbecauseofitshi#sensitivity，easymanipulation，hi#efficiencyinenrichingmexpressedgenesandnonnallzationoftargetedfrag-ments．Intheprocessofexperiment．theregularoccuiTencesoflossof矾expressedgenes(m

5、RNAsandtheircorrespondingeDNAs)andlowefficiencyinligationb6／t-wb-*nlargeeDNAfragmentsandplasmidsusuallyresllltedinbadlibrary．Inthepresentedstudy，someimprovedtechniqueswereintroducedintoexperimentsandtheireffectsevaluated，including(i)deliberateextraction，whichincreasedtheyieldoftar-getf

6、ractionationsbyatle鹅t30％；(ii)removalofphenolsandpolysaccharidesfromRNAthroughspecificallyextractionwithpolyvinylpoly‘pyrrolidone(PVPP)andhighcqn．centrationofKAca8well舶precipitationwithLiCI，whichincreasedconcentrationandspeciesofmRNAs，andresultedinanear3foldincreaseofthecorrespondingUni

7、-ESTs；(iii)purificationandfraetionationeDNAstlu-cughgelelectrophoresis，whichincreasedtheabundanceinmmscriptsandtheratiooflargeeDNAinserters(>500ho)．Duetotheaboveme&lsui'cs，thequalityofconstructedSSHlibraryw∞imprevedobviously．Keywords：Suppressionsubtractivehybridization；Library；Improv