Two-dimensional Capillary Electrophoresis - SELEX

描述(申请人提供)：摘要。需要高通量的适体产物用于诊断和基础研究。目前的技术是基于SELEX的，很难自动化，不适合高通量适配子的产生。最近，明尼苏达大学的鲍瑟和约克大学的克里洛夫开发了一种基于毛细管电泳法的快速适配子生成方法。他们的技术被Bowser称为毛细管电泳-SELEX(CE-SELEX)，基于观察到单链DNA在自由溶液中的迁移率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。在毛细管电泳法中，未复合的DNA文库以紧带的形式迁移。相比之下，DNA靶标复合体的迁移具有不同的迁移率。通过丢弃未复合的DNA并扩增与靶标复合的DNA，可以在一到四个选择循环中产生高亲和力的适体。我们建议开发一种仪器，将CE-SELEX的速度提高两个数量级。我们的系统基于二维毛细管电泳法，其中蛋白质样本在第一毛细管中被分离。在第一毛细管中可以分离多达100个组分。组分被自动转移到孵化器，在那里它们与寡核苷酸文库混合并可以与之复合。然后每个组分被转移到第二个毛细管中，在那里内容物经过CE-SELEX。未复合的DNA被丢弃，蛋白质复合体中的DNA被扩增。从二维系统的每个循环中扩增的DNA被汇集在一起，作为后续SELEX步骤的DNA文库。SELEX完成后，对产物DNA进行克隆和测序，并对相应的目的蛋白进行消化和质谱仪鉴定。 公共卫生相关性：(申请人提供)：说明。适配子是合成的DNA片段，与特定的靶点紧密结合。它们在靶标的分析中很有用，并已被用作治疗学。使用目前的技术，他们的生成速度很慢。这项提议将使适配子的生成速度提高两个数量级。

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. PUBLIC HEALTH RELEVANCE: (provided by applicant): Narrative. Aptamers are synthetic pieces of DNA that tightly bind to a specific target. They are useful in the analysis of the target and have found application as therapeutics. Using current technology, their generation is slow. This proposal will speed aptamer generation by two orders of magnitude.