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1、萬方數(shù)據(jù)2011年24卷5期VoL24No．5西南農(nóng)衛(wèi)學(xué)報Southm∞tChinaJournalofA面culturalSciences，1849文章編號：1001—4829(2011)05—1849—04幾個提高抑審IJ消減雜交(SSH)文庫質(zhì)量的技術(shù)改進(jìn)董登峰1’。，李楊瑞“，白云彩1(1．廣西大學(xué)農(nóng)學(xué)院，廣西南寧530004；2．廣西作物遺傳改良生物技術(shù)重點(diǎn)實驗室，廣西南寧530007)摘要：抑制消減雜交(SSH)高靈敏、易操作、高效富集低表達(dá)豐度基因和均一化目的基因片斷，廣泛用干差異表達(dá)基因的分離和eDNA文庫構(gòu)建。在實驗操作中常出現(xiàn)低表達(dá)mRNA和eDN
2、A丟失、大片斷連接效率低等問題，構(gòu)建的文庫質(zhì)量差，文章通過改進(jìn)萃取操作提高目的產(chǎn)物(mRNA和eDNA)回收率，回收率增加30％以上；通過采用PvPP、高濃度KAc專一性結(jié)合和LICl沉淀等措施除去RNA中的多酚和多糖、進(jìn)而增加可分離mRNA種類和含量，對應(yīng)的Uni-EST種類增加近3倍，通過采用膠回收并分段連接保護(hù)大片斷eDNA等措施使文庫中轉(zhuǎn)錄本豐富度增加，大片段(500bp以上)比例增大，eDNA平均長度增大，文庫質(zhì)量明顯提高。關(guān)鍵詞：抑制消減雜交(SSH)；文庫構(gòu)建；技術(shù)改進(jìn)中圖分類號：S181文獻(xiàn)標(biāo)識碼：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礬expressedgenes(m
5、RNAsandtheircorrespondingeDNAs)andlowefficiencyinligationb6／t-wb-*nlargeeDNAfragmentsandplasmidsusuallyresllltedinbadlibrary．Inthepresentedstudy，someimprovedtechniqueswereintroducedintoexperimentsandtheireffectsevaluated，including(i)deliberateextraction，whichincreasedtheyieldoftar-getf
6、ractionationsbyatle鵝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
