Rapid Electrophoretic Sorting of DNA using Nanoemulsion Tags

使用纳米乳液标签对 DNA 进行快速电泳分选

• 批准号: 0932536
• 负责人: James Schneider
• 金额: $ 33万
• 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: 2009
• 资助国家: 美国
• 起止时间: 2009-08-01 至 2012-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0932536&HistoricalAwards=false
• 关键词: Rapid; Electrophoretic; Sorting; DNA; using

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.

0932536SCHNEIDER“该奖项是根据2009年的《美国回收与再投资法》（公法111-5）资助的。为了鼓励DNA与纳米乳液液滴的相互作用，DNA通过各种非极性组进行了修饰。然后，该烷基化DNA（ADNA）可以用作标准的Sanger DNA测序过程的底漆。使用毛细管电泳（CE）进行分离，毛细管电泳是一种用于多种类型的生物分子表征和DNA测序的标准车辆。由于电泳分离不需要将聚合物或凝胶用作筛分矩阵，因此我们期望提供的运行时间比当前最新的毛细管电泳（ CGE）。此外，可以分析更长的DNA，从而大大降低了在Sanger过程中必须执行的酶促作品。最后，使用这些稀释表面活性剂溶液避免将粘性聚合物溶液引入毛细血管或微通道时遇到困难。智力优点：提出的过程是标记为游离溶液电泳（ELFSE）的末端的扩展，这是一种现有的DNA分离技术，可将未充电的聚合物或蛋白质阻力标签连接到DNA群体，以使其游离溶液迁移率是DNA长度的函数。尽管这将消除筛分矩阵的要求，但Elfse方法与CGE没有竞争力，因为必须过时单分散标签才能有效。在此系统中，ADNA和液滴或胶束之间的相互作用频繁且短暂寿命，因此每个ADNA掉期在运行过程中会标记数百万次。即使液滴可能是多分散的，这也赋予了A DNA上高度均匀的平均阻力。在运行期间，标签也可以按照给定长度的DNA分离所需的要求进行交换。该研究计划涉及开发与电泳兼容的纳米乳液制备方法，量化和最大化高电场中的峰值分辨率，并在运行过程中建立切换标签大小的方法以进行更大的测序吞吐量。更广泛的影响：DNA测序是一种非常有价值的生物分析方法，它彻底改变了对生物过程，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，并使它们在更广泛的问题上承受。对电场中溶解化和液滴流体动力学动力学的见解也将从工作中揭示。 DNA电泳是现代生物学实验室中普遍存在的过程，该方法也可能对其他领域产生很大的影响。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，该研究生专注于胶体表征方法。最后，他们将为少数民族学生（SAMS）开发一系列夏季学院的示威游行，这使少数民族高中生接触了科学和工程学的机会。该演示将涉及使用PCR方法来量化未知样品中的DNA量。