Two-dimensional Capillary Electrophoresis - SELEX
二维毛细管电泳 - SELEX
基本信息
- 批准号:8286819
- 负责人:
- 金额:$ 18.75万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:2011
- 资助国家:美国
- 起止时间:2011-06-18 至 2014-04-30
- 项目状态:已结题
- 来源:
- 关键词:AffinityAmino Acid SequenceAntibodiesBasic ScienceBindingBlood capillariesCapillary ElectrophoresisCloningColorComplexComplex MixturesContinuous InfusionDNADNA BindingDNA LibraryDNA-Binding ProteinsDepositionDiagnostics ResearchDigestionEquilibriumEvolutionFluorescenceFluorescent ProbesGenerationsHumanImmobilizationIncubatedIncubatorsLabelLasersLibrariesLigandsMass Spectrum AnalysisMethodsMinnesotaMonitorNomenclatureOligonucleotidesPeptide Sequence DeterminationPeptidesProceduresProcessPropertyProteinsProteomeRNAReproducibilityResolutionSamplingSingle-Stranded DNASolutionsSpeedSystemTechniquesTechnologyTherapeuticTimeTrypsinUniversitiesabstractingaptamerbasecapillarycomparativedetectorexperienceinstrumentmagnetic beadsmass spectrometermigrationprotein complexprotein expressionresponsetwo-dimensional
项目摘要
DESCRIPTION (provided by applicant): Abstract. There is a need for the high throughput generation of aptamers for diagnostic and basic research. Current technology is based on SELEX, which is very difficult to automate and is not suitable for high-throughput aptamer generation. Recently, Bowser at the University of Minnesota and Krylov at York University developed a high-speed method for aptamer generation based on capillary electrophoresis. Their technology, which Bowser calls capillary electrophoresis-SELEX (CE-SELEX), is based on the observation that the mobility of single stranded DNA in free solution is independent of the oligonucleotide's sequence. In CE-SELEX, a target molecule is incubated with an oligonucleotide library. The uncomplexed DNA library migrates as a tight band during capillary electrophoresis. In contrast, DNA-target complexes migrate with a different mobility. By discarding the uncomplexed DNA and amplifying the DNA that is complexed with the target, high affinity aptamers can be generated in one to four selection cycles. We propose to develop an instrument that will increase the speed of CE- SELEX by two orders of magnitude. Our system is based on two-dimensional capillary electrophoresis, where a protein sample is separated in the first capillary. Up to 100 components can be separated in the first capillary. Fractions are automatically transferred to an incubator, where they are mixed with and can complex with an oligonucleotide library. Each fraction is then transferred to a second capillary, where the contents undergo CE-SELEX. Uncomplexed DNA is discarded and the DNA in the protein-complexes is amplified. The amplified DNA from each cycle of the two-dimensional system is pooled and used as the DNA library for subsequent SELEX steps. After SELEX is complete, the product DNA is cloned and sequenced, and the corresponding target protein is digested and identified by mass spectrometry.
描述(由申请人提供):摘要。诊断和基础研究需要高通量的适体生成。目前的技术是基于SELEX的,这是非常难以自动化的,不适合高通量适配体的生成。最近,明尼苏达大学的Bowser和约克大学的Krylov开发了一种基于毛细管电泳的快速适配体生成方法。他们的技术,Bowser称之为毛细管电泳- selex (CE-SELEX),是基于观察到单链DNA在自由溶液中的流动性与寡核苷酸的序列无关。在CE-SELEX中,目标分子与寡核苷酸文库孵育。在毛细管电泳过程中,不复杂的DNA文库作为一个紧带迁移。相反,dna靶复合物以不同的移动性迁移。通过丢弃未络合的DNA并扩增与目标络合的DNA,高亲和力的适体可以在一到四个选择周期内产生。我们建议开发一种仪器,将CE- SELEX的速度提高两个数量级。我们的系统是基于二维毛细管电泳,其中蛋白质样品在第一毛细管分离。在第一根毛细管中可分离多达100个组分。分数被自动转移到培养箱中,在那里它们与寡核苷酸库混合并可以与寡核苷酸库配合。然后将每个馏分转移到第二个毛细管,其中的内容物进行CE-SELEX。未合成的DNA被丢弃,蛋白质复合物中的DNA被扩增。从二维系统的每个周期扩增的DNA被汇集并用作后续SELEX步骤的DNA文库。SELEX完成后,对产物DNA进行克隆和测序,并对相应的靶蛋白进行酶切和质谱鉴定。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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Norman J Dovichi其他文献
Climbing the protein ladder
爬上蛋白质阶梯
- DOI:
10.1038/nbt1004-1242 - 发表时间:
2004-10-01 - 期刊:
- 影响因子:41.700
- 作者:
Norman J Dovichi - 通讯作者:
Norman J Dovichi
Norman J Dovichi的其他文献
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{{ truncateString('Norman J Dovichi', 18)}}的其他基金
Quantitative proteomics of Xenopus oocytes, eggs, blastomeres, and nuclear transplants
非洲爪蟾卵母细胞、卵子、卵裂球和核移植物的定量蛋白质组学
- 批准号:
8937359 - 财政年份:2015
- 资助金额:
$ 18.75万 - 项目类别:
Quantitative proteomics of Xenopus oocytes, eggs, blastomeres, and nuclear transplants
非洲爪蟾卵母细胞、卵子、卵裂球和核移植物的定量蛋白质组学
- 批准号:
9116239 - 财政年份:2015
- 资助金额:
$ 18.75万 - 项目类别:
Two-dimensional Capillary Electrophoresis - SELEX
二维毛细管电泳 - SELEX
- 批准号:
8453389 - 财政年份:2011
- 资助金额:
$ 18.75万 - 项目类别:
Diagonal Capillary Electrophoresis for Phosphorylation and Sialylation Analysis
用于磷酸化和唾液酸化分析的对角毛细管电泳
- 批准号:
8710257 - 财政年份:2011
- 资助金额:
$ 18.75万 - 项目类别:
Technology for single cell proteomics and its application to the analysis of single Xenopus blastomeres
单细胞蛋白质组学技术及其在非洲爪蟾单个卵裂球分析中的应用
- 批准号:
9198018 - 财政年份:2011
- 资助金额:
$ 18.75万 - 项目类别:
Two-dimensional Capillary Electrophoresis - SELEX
二维毛细管电泳 - SELEX
- 批准号:
8164257 - 财政年份:2011
- 资助金额:
$ 18.75万 - 项目类别:
Diagonal Capillary Electrophoresis for Phosphorylation and Sialylation Analysis
用于磷酸化和唾液酸化分析的对角毛细管电泳
- 批准号:
8306043 - 财政年份:2011
- 资助金额:
$ 18.75万 - 项目类别:
Diagonal Capillary Electrophoresis for Phosphorylation and Sialylation Analysis
用于磷酸化和唾液酸化分析的对角毛细管电泳
- 批准号:
8534195 - 财政年份:2011
- 资助金额:
$ 18.75万 - 项目类别:
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