Rapid purification following enzyme-catalyzed nucleic acid reactions

描述(由申请人提供)：在DNA分析和操作中使用的生化反应对包括取证、诊断基因测试和生物医学研究在内的许多应用程序都很重要。切割DNA并将其插入克隆位置的酶反应是疾病研究和利用微生物表达具有医疗价值的重要蛋白质的许多策略的基础。为了获得所需的反应成分，同时丢弃那些可能干扰DNA进一步使用的成分，纯化是必不可少的，这对基因组学所涉及的时间、成本和劳动力都有很大的贡献。改进工作流程，并在下一代测序的文库生成过程中实现清理步骤的自动化，将消除基因组图谱这项令人兴奋且前景光明的技术中的瓶颈之一。迪菲尼蒂基因公司已获得罗切斯特大学PI实验室开发的新型材料技术的许可，该技术可用于提纯核酸反应所需的快速、廉价和简单的生物分子分离。特别是，Diffity发布了一种产品，能够在测序反应之前进行聚合酶链扩增后快速、高效地提纯DNA。本提案中的工作涉及对该技术进行调整，以生产用于酶反应快速纯化的产品。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，然后选择性地重新溶解所需的组分。Diffity的提纯方法是不同的，因为它使用特殊配置的表面，吸收溶液中不需要的成分，而将需要的成分留在溶液中。这使得可以通过将具有特殊功能化表面的颗粒保留在吸管尖端来实现单步过程，从而将纯化过程简化为简单地吸入反应溶液并分配纯化的DNA反应溶液。我们将证明，我们可以配置适当的二氧化硅颗粒表面，在酶反应后一步60秒提取不需要的成分，同时将需要的成分留在溶液中。 公共卫生相关性：酶催化的DNA反应被广泛用于医疗保健应用，如诊断、病原体检测和出于治疗目的的克隆。这些反应通常需要进行昂贵且劳动密集型的提纯。我们建议制造含有新型过滤材料的功能性吸管尖端，这些材料可用于显著减少与当前净化方案相关的时间、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.