Two-dimensional Capillary Electrophoresis - SELEX

二维毛细管电泳 - SELEX

基本信息

批准号：
8164257
负责人：
Norman J Dovichi
金额：
$ 18.25万
依托单位：
UNIVERSITY OF NOTRE DAME
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：
https://reporter.nih.gov/project-details/8164257
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

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. 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.

描述（由申请人提供）：摘要。需要高吞吐量的适体用于诊断和基础研究。当前的技术基于SELEX，这很难自动化，并且不适合高通量适体生成。最近，明尼苏达大学和约克大学Krylov的Bowser开发了一种基于毛细管电泳的适体生成的高速方法。他们的技术称为毛细血管电泳 - 塞莱克斯（CE-SELEX），基于这样的观察结果，即自由溶液中单链DNA的迁移率与寡核苷酸的序列无关。在CE-SELEX中，将目标分子与寡核苷酸库一起孵育。未复杂的DNA文库在毛细管电泳过程中以紧密的条带迁移。相反，DNA目标复合物以不同的迁移率迁移。通过丢弃未复杂的DNA并扩增与靶标复合的DNA，可以在一到四个选择周期中产生高亲和力适体。我们建议开发一种将使CE-SELEX速度提高两个数量级的仪器。我们的系统基于二维毛细管电泳，其中蛋白质样品在第一个毛细管中分离。在第一个毛细管中可以分开多达100个组件。分数会自动转移到孵化器中，并将其与寡核苷酸库混合并可以复杂化。然后将每个部分转移到第二个毛细管中，其中内容物在其中进行CE-SELEX。丢弃未复合的DNA，并放大蛋白质复合物中的DNA。从二维系统的每个循环中的扩增DNA汇总，并用作随后的SELEX步骤的DNA库。 SELEX完成后，将产物DNA克隆并测序，并通过质谱法消化和鉴定相应的靶蛋白。公共卫生相关性：（申请人提供）：叙述。适体是与特定靶标紧密结合的DNA的合成部分。它们在分析目标的分析中很有用，并发现了作为治疗剂的应用。使用当前的技术，他们的一代很慢。该提案将加快适sa剂的生成两个数量级。