Rapid, Free-Solution Electrophoretic Separations of Kilobase DNA by Transiently Attached Wormlike Micelles

通过瞬时附着的蠕虫状胶束对千碱基 DNA 进行快速、自由溶液电泳分离

• 批准号: 1605351
• 负责人: James Schneider
• 金额: $ 29.54万
• 依托单位: Carnegie-Mellon University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-08-01 至 2019-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1605351&HistoricalAwards=false
• 关键词: Rapid; Free; Solution; Electrophoretic; Separations

Proposal Number: 1605351, ScheiderRapid, Free-Solution Electrophoretic Separations of Kilobase DNA by Transiently Attached MicellesThis project will develop an ultrafast method to analyze long-strand DNA without the use of gels but with shorter analysis times. Many important DNA sequencing methods require a length-based sorting of DNA longer than 1000-10,000 bases. The most common method to accomplish this is pulsed-field gel electrophoresis, which requires 12-36 hours to complete and presents a significant bottleneck. The proposed method instead uses low viscosity, water-based solutions of surfactants instead of gels, and is expected to perform the same function as mentioned in gel electrophoresis but with runtimes of less than five minutes. Additionally, the low-viscosity solutions used are easy to inject into capillaries and microchips and do not suffer from clogging and degradation with repeated use. Incorporation of this rapid, gel-free method into critical DNA sequencing procedures could greatly decrease the cost of sequencing methods used in forensic identification and biology research such as genome mapping and pathogen identification. One Ph.D student, three M.S. students, and five undergraduates will be trained in colloid and interface science, organic chemistry, and bioseparations as they contribute to the project. Single-molecule DNA visualization studies will be built into demonstrations for K-12 student outreach, and lab modules for undergraduate courses will be developed based on microchip electrophoresis.This project will research a rapid, non-gel means to separate double strand DNA greater than 10-100 kilobases (kB) in length as required for genome mapping and other applications using inexpensive, widely available capillary electrophoresis equipment. The method uses transiently attached wormlike micelles as drag-tags, which, when attached to DNA by a terminal n-alkane group, provide a length-dependent electrophoretic mobility without the use of a sieving matrix. Specific objectives of the project aims are: (1) to design and implement substituted peptide nucleic acid staples to attach alkyl groups to the ends of 48.5 kB lambda-DNA and its digests; (2) to screen a series of nonionic micelle buffers to obtain the largest drag-tag size possible; (3) to develop a theory predicting the mobility of the DNA by consideration of DNA-drag-tag segregation effects in networks of varying pore size and micelle lifetimes; and (4) to perform single-molecule imaging of DNA to assess the extent of DNA-drag-tag segregation. The proposed work will identify optimal buffers and run conditions for a given set of DNA lengths to be separated and identify limits of DNA length and runtime that can be achieved. Insights into the perturbation of wormlike micelle entanglements by vicinal hydrophobes may also help better understand the rheological behavior of these solutions in the presence of contaminants.

提案编号：1605351，ScheiderRapid，通过瞬时附着胶束的千碱基DNA的自由溶液电泳分离该项目将开发一种超快方法来分析长链DNA，而不使用凝胶，但分析时间更短。许多重要的DNA测序方法需要对长于1000- 10，000个碱基的DNA进行基于长度的分选。最常用的方法是脉冲场凝胶电泳，需要12-36小时才能完成，这是一个很大的瓶颈。所提出的方法使用低粘度的表面活性剂水基溶液代替凝胶，并且预期执行与凝胶电泳中所述相同的功能，但运行时间小于5分钟。此外，所用的低粘度溶液易于注入毛细管和微芯片中，并且在重复使用时不会发生堵塞和降解。将这种快速、无凝胶的方法结合到关键的DNA测序程序中，可以大大降低用于法医鉴定和生物学研究（如基因组作图和病原体鉴定）的测序方法的成本。一个博士生，三个硕士生。学生和五名本科生将接受胶体和界面科学，有机化学和生物分离方面的培训，因为他们为该项目做出了贡献。单分子DNA可视化研究将被纳入K-12学生推广的演示中，并将基于微芯片电泳开发本科课程的实验模块。该项目将研究一种快速，非凝胶方法，以分离长度超过10-100 kB的双链DNA，以满足基因组作图和其他应用的需要，使用廉价，广泛使用的毛细管电泳设备。该方法使用瞬时附着的蠕虫状胶束作为拖曳标签，当通过末端正烷烃基团附着于DNA时，其提供长度依赖性电泳迁移率而不使用筛分基质。该项目的具体目标是：（1）设计和实现取代的肽核酸斯台普斯，将烷基连接到48.5kB的DNA及其钉合物的末端;（2）筛选一系列非离子胶束缓冲液，以获得尽可能大的拖曳标签尺寸;（3）通过考虑不同孔径和胶束寿命的网络中的DNA-拖曳-标签分离效应，开发预测DNA的移动性的理论;和（4）进行DNA的单分子成像以评估DNA-药物-标签分离的程度。拟议的工作将确定最佳的缓冲液和运行条件，为一组给定的DNA长度被分离，并确定可以实现的DNA长度和运行时间的限制。通过邻位疏水物对蠕虫状胶束缠结的扰动的了解也可能有助于更好地理解这些溶液在污染物存在下的流变行为。