Rapid purification following enzyme-catalyzed nucleic acid reactions

描述（由申请人提供）：DNA分析和操作中使用的生化反应对于许多应用都很重要，包括法医学、诊断性基因检测和生物医学研究。将DNA切割和插入克隆位点的酶促反应是疾病研究和利用微生物表达具有医学价值的重要蛋白质的许多策略的基础。纯化以获得所需的反应组分，同时丢弃那些可能干扰DNA进一步使用的组分是必要的，并且对基因组学中涉及的时间、成本和劳动力有显著贡献。改进工作流程并在下一代测序的文库生成期间实现清理步骤的自动化将消除基因组作图的令人兴奋和有前途的技术中的瓶颈之一。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。特别是，Diffinity已经发布了一种能够在测序反应之前的聚合酶链扩增之后快速、有效地纯化DNA的产品。本提案中的工作涉及调整该技术以制备用于酶促反应的快速纯化的产品。目前纯化这些反应的方法需要多个步骤，并使用试剂将溶液中的所有生物分子结合到底物上，然后选择性地再溶解所需的组分。Diffinity的净化方法与众不同，因为它使用特殊配置的表面，吸引溶液中不需要的成分，同时将所需的成分留在溶液中。这使得能够实现单步过程，该单步过程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。我们将证明，我们可以配置二氧化硅颗粒表面适合于一步，60秒提取酶促反应后的不需要的组分，而留下所需的溶液中。 公共卫生相关性：酶催化的DNA反应广泛用于医疗保健应用，例如诊断、病原体检测和用于治疗目的的克隆。这些反应通常需要昂贵且劳动密集的纯化。我们建议制作含有新型过滤材料的功能性移液器吸头，这些过滤材料可用于显著减少与当前纯化方案相关的时间、成本和环境浪费。

DESCRIPTION (provided by applicant): Biochemical reactions used in the analysis and manipulation of DNA are important for many applications including forensics, diagnostic genetic testing and biomedical research. Enzymatic reactions to cut and insert DNA into cloning sites underpin many strategies for disease research and for use of microorganisms to express important proteins with medical value. Purification to obtain the desired reaction components while discarding those that can interfere with further use of the DNA is essential and comprises a significant contribution to the time, cost and labor involved in genomics. Improving workflow and enabling automation in the cleanup steps during library generation for next generation sequencing would remove one of the bottlenecks in that exciting and promising technology for genomic mapping. 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. In particular, Diffinity has released a product enabling rapid, efficient purification of DNA after polymerase chain amplification prior to sequencing reactions. The work in the present proposal involves adapting that technology to make products for rapid purification of enzymatic reactions. Current approaches to purifying these reactions require multiple steps and use reagents to bind all of the biomolecules in solution to a substrate and then selectively redissolve the desired component. Diffinity's purification method is distinct because it uses specially configured surfaces that attract undesired components of a solution while leaving the desired ones in solution. This enables a single-step process 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. We will demonstrate that we can configure silica particle surfaces appropriate to one-step, 60 second extraction of the undesired components following enzymatic reactions while leaving the desired ones in solution. PUBLIC HEALTH RELEVANCE: Enzyme-catalyzed DNA reactions are widely used in health care applications such as diagnosis, pathogen detection and cloning for therapeutic purposes. These reactions routinely require purification that is costly and labor-intensive. We propose to make functional pipette tips containing novel filtration materials that can be used to dramatically reduce the time, cost and environmental waste associated with current purification protocols.