Rapid DNA extraction from gels

从凝胶中快速提取 DNA

• 批准号: 8199999
• 负责人: LEWIS J ROTHBERG
• 金额: $ 10.16万
• 依托单位: DIFFINITY GENOMICS, INC.
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-01 至 2012-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8199999
• 关键词: Adsorption; Aspirate substance; Automobile Driving; Back; Binding; Buffers; Centrifugation; Cloning; Competitive Binding; DNA; DNA Binding; DNA Sequence; Diagnosis; Digestion; Electrophoresis; Equilibrium; Ethanol; Fluorescence; Gel; Genetic; Genomics; Goals; Homidium Bromide; Individual; Intercalating Agents; Left; Licensing; Link; Liquid substance; Measurement; Medical Research; Medicine; Methods; Metric; Molecular; Nucleic Acids; Phase; Plant Resins; Process; Protocols documentation; Reaction; Reagent; Recovery; Relative (related person); Sampling; Sepharose; Silicon Dioxide; Slice; Sodium Chloride; Solutions; Source; Speed; Staining method; Stains; Surface; Technology; Therapeutic; Time; Tube; Universities; Water; Work; base; cost; design; ds-DNA; follow-up; improved; intercalation; interest; next generation; novel; novel strategies; particle; restriction enzyme; success; translational study

DESCRIPTION (provided by applicant): Extraction of DNA from gels following electrophoresis is widely used to obtain purified size selected DNA fragments from mixtures. The DNA thus obtained is used for cloning and sequencing reactions. Gel extraction is part of next generation sequencing protocols that are becoming the primary source of genetic information. That information is widely used in translational studies and promises to be a cornerstone of "personalized medicine" where therapeutic courses are linked to an individual's genetic makeup. Rapid and simple nucleic purification products that improve workflow and reduce cost are vital to advances in genomics. Diffinity Genomics has licensed novel materials technology developed in the PI's lab at the University of Rochester that can be used for fast, inexpensive and simple biomolecular separations needed to purify nucleic acid reactions. The work in the present proposal involves adapting that technology and adding a new approach to selective capture of DNA from dissolved gel slices using silica particles functionalized with DNA intercalators. Our goal is to make products for rapid gel extraction using a three step pipette-based protocol. Current approaches to extraction of DNA from gels require multiple steps taking more than 10 minutes and use several reagents. Diffinity's purification methods are distinct because they use specially configured surfaces that selectively adsorb or repel desired solution components. This enables a process with fewer steps that can be implemented by retaining particles with the specially functionalized surfaces in pipette tips so that the purification process is reduced simply to aspirating the reaction solution and dispensing the purified DNA reaction solution. The approach based on functional pipettes is easily automated with liquid handlers for high throughput applications. We will demonstrate that we can configure silica particle surfaces appropriate to a three-step extraction of DNA from dissolved gel slices that take less than 3 minutes. If successful, in Phase II, we will integrate the particles into pipette tips designed to enable rapid mixing, verify the efficacy for applications to DNA sequencing and cloning, and exploit the manufacturing technology developed for a current product (RapidTipTM) to launch a gel extraction product. PUBLIC HEALTH RELEVANCE: Cloning and sequencing of DNA are very important to medical research and diagnosis. Extracting DNA from gels following electrophoresis is widely used for cloning applications and is part of next generation sequencing protocols. We propose a new approach to extracting DNA from gels that will reduce the time; cost and labor associated with the process and be easily automated for high throughput applications.

描述（由申请人提供）：电泳后从凝胶中提取DNA被广泛用于从混合物中获得纯化的大小选择的DNA片段。由此获得的DNA用于克隆和测序反应。凝胶提取是下一代测序方案的一部分，正在成为遗传信息的主要来源。这些信息被广泛用于转化研究，并有望成为“个性化医疗”的基石，其中治疗课程与个人的基因组成有关。快速和简单的核酸纯化产品，改善工作流程和降低成本是至关重要的基因组学的进步。Diffinity Genomics公司已授权罗切斯特大学PI实验室开发的新材料技术，可用于纯化核酸反应所需的快速、廉价和简单的生物分子分离。本提案中的工作涉及调整该技术，并增加一种新的方法，使用DNA嵌入剂功能化的二氧化硅颗粒从溶解的凝胶切片中选择性捕获DNA。我们的目标是使用基于移液器的三步方案制造用于快速凝胶提取的产品。目前从凝胶中提取DNA的方法需要花费超过10分钟的多个步骤，并使用几种试剂。Diffinity的净化方法与众不同，因为它们使用特殊配置的表面，选择性地吸附或排斥所需的溶液成分。这使得能够实现具有更少步骤的方法，其可以通过将具有特殊功能化表面的颗粒保留在移液管尖端中来实现，使得纯化过程简单地减少为抽吸反应溶液和分配纯化的DNA反应溶液。基于功能移液器的方法很容易通过液体处理器实现自动化，以实现高通量应用。我们将证明，我们可以配置二氧化硅颗粒表面适合于从溶解的凝胶切片中提取DNA的三步提取，这需要不到3分钟。如果成功，在第二阶段，我们将把颗粒整合到移液器吸头中，以实现快速混合，验证DNA测序和克隆应用的有效性，并利用为当前产品开发的制造技术（RapidTipTM）推出凝胶提取产品。 公共卫生相关性：DNA的克隆和测序对医学研究和诊断非常重要。电泳后从凝胶中提取DNA广泛用于克隆应用，并且是下一代测序方案的一部分。我们提出了一种从凝胶中提取DNA的新方法，该方法将减少与该过程相关的时间、成本和劳动力，并且易于自动化用于高通量应用。