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Two-dimensional Capillary Electrophoresis - SELEX

二维毛细管电泳 - SELEX

基本信息

批准号：
8453389
负责人：
Norman J Dovichi
金额：
$ 18.09万
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/8453389
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.

描述（由申请人提供）：摘要。诊断和基础研究需要高通量产生适体。目前的技术基于SELEX，很难实现自动化，并且不适合高通量适配体生成。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适配体高速生成方法。 Bowser 将他们的技术称为毛细管电泳-SELEX (CE-SELEX)，该技术基于这样的观察：游离溶液中单链 DNA 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一起孵育。未复合的 DNA 文库在毛细管电泳过程中作为紧密条带迁移。相比之下，DNA-靶标复合物以不同的迁移率迁移。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周期内生成高亲和力适体。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，其中蛋白质样品在第一个毛细管中分离。第一个毛细管中最多可分离 100 个组分。级分被自动转移到培养箱，在那里它们与寡核苷酸文库混合并可以与寡核苷酸文库复合。然后将每个级分转移至第二个毛细管，其中的内容物在此进行 CE-SELEX。未复合的 DNA 被丢弃，蛋白质复合物中的 DNA 被扩增。来自二维系统每个循环的扩增 DNA 被汇集并用作后续 SELEX 步骤的 DNA 库。 SELEX完成后，对产物DNA进行克隆和测序，并消化并通过质谱鉴定相应的目标蛋白。

项目成果

期刊论文数量（4）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Nicked-sleeve interface for two-dimensional capillary electrophoresis.

DOI：
10.1039/c3an00284e
发表时间：
2013-07-07
期刊：
The Analyst
影响因子：
0
作者：
Flaherty RJ;Huge BJ;Bruce SM;Dada OO;Dovichi NJ
通讯作者：
Dovichi NJ

High efficiency and quantitatively reproducible protein digestion by trypsin-immobilized magnetic microspheres.

DOI：
10.1016/j.chroma.2011.11.050
发表时间：
2012-01-13
期刊：
Journal of chromatography. A
影响因子：
0
作者：
Sun L;Li Y;Yang P;Zhu G;Dovichi NJ
通讯作者：
Dovichi NJ

Capillary electrophoresis coupled with automated fraction collection.

DOI：
10.1016/j.talanta.2014.07.018
发表时间：
2014-12
期刊：
TALANTA
影响因子：
6.1
作者：
Huge, Bonnie Jaskowski;Flaherty, Ryan J.;Dada, Oluwatosin O.;Dovichi, Norman J.
通讯作者：
Dovichi, Norman J.