Rapid Electrophoretic Sorting of DNA using Nanoemulsion Tags

0932536 Schneider“该奖项是根据2009年美国复苏和再投资法案（公法111-5）资助的。研究人员提出了一种快速DNA电泳的新方法，该方法使用纳米乳液液滴的稀释悬浮液，这些液滴瞬时结合样品中的DNA。为了促进DNA与纳米乳液液滴的相互作用，用各种非极性基团对DNA进行末端修饰。然后，该烷基化DNA（aDNA）可用作标准桑格DNA测序方法的引物。使用毛细管电泳（CE）进行分离，毛细管电泳是许多类型的生物分子表征和DNA测序的标准载体。由于电泳分离不需要使用聚合物或凝胶作为筛分基质，我们期望提供比现有技术的毛细管凝胶电泳（CGE）快10-100倍的运行时间。此外，可以分析更长的DNA，大大减少了桑格法中必须进行的酶促工作。最后，使用这些稀释的表面活性剂溶液避免了在将粘性聚合物溶液引入毛细管或微通道中时遇到的困难。智力优势：所提出的方法是末端标记自由溶液电泳（ELFSE）的延伸，ELFSE是一种现有的DNA分离技术，其将不带电的聚合物或蛋白质拖拽标签共价连接到DNA群体，使得它们的自由溶液迁移率是DNA长度的函数。虽然这将消除对筛分基质的要求，但ELFSE方法与CGE没有竞争力，因为标签需要禁止单分散才能有效。在这个系统中，aDNA与液滴或胶束之间的相互作用频繁且短暂，因此每个aDNA在运行期间交换标签数百万次。这赋予α DNA高度均匀的平均阻力，即使液滴可能是多分散的。标签也可以在运行期间根据需要交换，以分离给定长度的DNA。该研究计划涉及开发与电泳兼容的纳米乳液制备方法，定量和最大化高电场中的峰分辨率，以及建立在运行期间切换标签大小以获得更大测序通量的方法。更广泛的影响：DNA测序是一种非常有价值的生物分析方法，它彻底改变了生物过程，微生物多样性和进化的研究。这种方法将大大降低这些方法的成本，并使它们对更广泛的问题产生影响。洞察力的动态溶解和液滴流体动力学在电场中也将揭示从工作。DNA电泳是现代生物学实验室中普遍存在的过程，该方法也可能在其他领域产生巨大影响。研究人员还将赞助几个本科生研究项目，并为研究生课程开发一个新的实验室，重点是胶体表征方法。最后，他们将为少数民族学生暑期学院（SAMS）开发一系列动手示范，使少数民族高中生获得科学和工程方面的机会。演示将涉及使用PCR方法来量化未知样本中的DNA量。

0932536Schneider"This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5)."The investigators propose a novel method of rapid DNA electrophoresis that uses dilute suspensions of nanoemulsion droplets that transient bind to DNA in the sample. To encourage interaction of the DNA with the nanoemulsion droplets, DNA is end modified with various nonpolar groups. This alkylated DNA (aDNA) can then be used as a primer for a standard Sanger DNA sequencing process. Separations are carried out using capillary electrophoresis (CE), a standard vehicle for many types of biomolecular characterization and DNA sequencing. Because the electrophoretic separation does not require the use of polymers or gels as a sieving matrix, we expect to provide run times that are 10-100 times faster than can be achieved by the current state-of-the-art, capillary gel electrophoresis (CGE). In addition, much longer DNA can be analyzed, greatly reducing the enzymatic work that must be performed in the Sanger process. Finally, difficulties encountered when introducing viscous polymer solutions into capillaries or microchannels are avoided using these dilute surfactant solutions. Intellectual Merit: The process proposed is an extension of end labeled free solution electrophoresis (ELFSE), an existing DNA separation technique that covalently attaches uncharged polymer or protein drag tags to DNA populations so that their free solution mobility is a function of DNA length. While this would remove the requirement of a sieving matrix, ELFSE methods have not been competitive with CGE as the tags need to be prohibitively monodisperse to be effective. In this system, interactions between aDNA and droplets or micelles are frequent and short lived, so that each aDNA swaps tags millions of times during the run. This confers a highly uniform average drag upon the a DNA, even though the droplets may be polydisperse. Tags can also be swapped during the run as required for separation of a given length of DNA. The research plan involves developing nanoemulsion preparation methods that are compatible with electrophoresis, quantifying and maximizing the peak resolution in high electric fields, and establishing means of switching tag sizes during the run for greater sequencing throughput. Broader Impacts: DNA sequencing is a tremendously valuable bioanalysis method that has revolutionized the study of biological processes, microbial diversity, and evolution. This method will dramatically reduce the cost of these methods and bring them to bear on a wider range of problems. Insights into the dynamics of solubilization and droplet hydrodynamics in electric fields will also be revealed from the work. DNA electrophoresis is a ubiquitous process in modern biology labs and the method is likely to have a large impact in other arenas as well. The investigators will also sponsor several undergraduate research projects and develop a new lab for a graduate course focused on methods of colloidal characterization. Finally, they will develop a series of hands on demonstrations for the Summer Academy for Minority Students (SAMS), which exposes minority high school students to opportunities in science and engineering. The demonstration will involve using PCR methods to quantify the amount of DNA in an unknown sample.