Two-dimensional Capillary Electrophoresis - SELEX

二维毛细管电泳 - SELEX

• 批准号: 8286819
• 负责人: Norman J Dovichi
• 金额: $ 18.75万
• 依托单位: UNIVERSITY OF NOTRE DAME
• 依托单位国家: 美国
• 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: https://reporter.nih.gov/project-details/8286819
• 关键词: Affinity; Amino Acid Sequence; Antibodies; Basic Science; Binding; Blood capillaries; Capillary Electrophoresis; Cloning; Color; Complex; Complex Mixtures; Continuous Infusion; DNA; DNA Binding; DNA Library; DNA-Binding Proteins; Deposition; Diagnostics Research; Digestion; Equilibrium; Evolution; Fluorescence; Fluorescent Probes; Generations; Human; Immobilization; Incubated; Incubators; Label; Lasers; Libraries; Ligands; Mass Spectrum Analysis; Methods; Minnesota; Monitor; Nomenclature; Oligonucleotides; Peptide Sequence Determination; Peptides; Procedures; Process; Property; Proteins; Proteome; RNA; Reproducibility; Resolution; Sampling; Single-Stranded DNA; Solutions; Speed; System; Techniques; Technology; Therapeutic; Time; Trypsin; Universities; abstracting; aptamer; base; capillary; comparative; detector; experience; instrument; magnetic beads; mass spectrometer; migration; protein complex; protein expression; response; two-dimensional

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。