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

该项目将开发一种超快速的方法来分析长链DNA，而不使用凝胶，但分析时间更短。许多重要的DNA测序方法需要对长度超过1000- 10000个碱基的DNA进行基于长度的排序。最常用的方法是脉冲场凝胶电泳，需要12-36小时才能完成，并且存在明显的瓶颈。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，并且有望实现与凝胶电泳相同的功能，但运行时间少于5分钟。此外，所使用的低粘度溶液易于注入毛细血管和微芯片，并且在重复使用时不会发生堵塞和降解。将这种快速、无凝胶的方法纳入关键的DNA测序程序中，可以大大降低法医鉴定和生物学研究（如基因组定位和病原体鉴定）中使用的测序方法的成本。一名博士生，三名硕士学生和五名本科生将在胶体和界面科学，有机化学和生物分离方面进行培训，因为他们对项目有所贡献。单分子DNA可视化研究将被建立在K-12学生推广的演示中，而基于微芯片电泳的本科课程的实验室模块将被开发。该项目将研究一种快速、非凝胶的方法，以分离长度大于10-100千碱基（kB）的双链DNA，用于基因组定位和其他应用，使用廉价、广泛可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标签，当通过末端正烷烃基团附着到DNA上时，提供长度依赖的电泳迁移率，而无需使用过筛基质。该项目的具体目标是：(1)设计并实现取代肽核酸钉，将烷基连接到48.5 kB的lambda-DNA及其酶切末端；(2)筛选一系列非离子胶束缓冲液，以获得尽可能大的拖标尺寸；(3)通过考虑不同孔径和胶束寿命的网络中DNA-拖-标签分离效应，建立预测DNA迁移的理论；(4)对DNA进行单分子成像，以评估DNA-标签分离的程度。所提出的工作将确定一组给定的DNA长度分离的最佳缓冲和运行条件，并确定可以实现的DNA长度和运行时间的限制。邻近疏水剂对蠕虫状胶束纠缠的扰动也有助于更好地理解这些溶液在污染物存在下的流变行为。

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.