Rapid intercalator removal from size-selected DNA for next generation sequencing

描述（由申请人提供）：来自下一代测序的信息广泛用于转化研究，并有望成为“个性化医学”的基石，其中治疗课程与个体的遗传组成有关。电泳后从凝胶中提取DNA以获得大小选择的DNA片段是所有主要的下一代测序平台的方案中存在的文库制备的劳动密集型部分。最近，Sage Science推出了一种用于DNA大小选择的颠覆性产品，该产品可以将所需的DNA片段从凝胶中“切换”出来，从而无需物理提取DNA。由于其速度和节省劳动力的协议，Sage Science Pippin Prep正在迅速被快速增长的下一代测序市场所采用。不幸的是，对于Pippin Prep，仍然需要进行相对劳动密集型的纯化以去除用于鉴定条带的嵌入染料，因为它们干扰下一代测序文库制备中的后续过程。Diffinity Genomics提出开发一种基于功能移液器吸头的单步纯化过程，该过程将在不到60秒的时间内消除嵌入染料，同时保留所需的DNA。 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。颗粒表面被特别配置为选择性地吸附或排斥各种溶液组分，使得可以在去除不需要的杂质的同时将所需组分保留在溶液中。差异利用了这一差异 采用快速吸附技术，推出快速PCR纯化产品（RapidTipTM），只需将PCR产物吸入功能性移液器吸头并分配纯化的DNA反应溶液，即可在不到一分钟的时间内完成一步PCR净化。本提案中的工作是开发具有表面的颗粒，所述表面选择性地结合所使用的嵌入染料， Sage科学，使颗粒易于分散在水中，以促进快速混合和收集杂质。我们还将确定用Sage Science仪器和Diffinity纯化获得的大小选择的DNA是否适用于下一代测序文库制备。 公共卫生相关性：下一代DNA测序正在迅速发展，因为它提供了对医学研究和诊断重要的遗传信息。使用凝胶电泳进行DNA大小选择是下一代测序方案的一部分，Sage Science的一种新产品正在简化这一过程。我们提出了一种配套产品，它将减少与纯化从Sage Science过程中获得的大小选择的DNA相关的时间、成本和材料浪费。

DESCRIPTION (provided by applicant): Information from next generation sequencing 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. Extraction of DNA from gels following electrophoresis to obtain size-selected DNA fragments is a labor intensive part of library preparation present in the protocols of all of the major next generation sequencing platforms. Recently, Sage Science introduced a disruptive product for DNA size selection that electrically "switches" the desired DNA fragments out of the gel and obviates the need for physically extracting the DNA. Due to its speed and labor saving protocol, the Sage Science Pippin Prep is being quickly adopted by the rapidly growing next generation sequencing market. Unfortunately, with the Pippin Prep it remains necessary to do a relatively labor-intensive purification to remove the intercalating dyes used to identify the bands because they interfere with subsequent processes in next generation sequencing library preparation. Diffinity Genomics proposes to develop a single-step purification process based on a functional pipette tip that will eliminate intercalating dyes in less than 60 seconds while retaining the desired DNA. 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. Particle surfaces are specially configured to selectively adsorb or repel various solution components making it possible to retain desired components in solution while removing unwanted impurities. Diffinity has utilized that differential adsorption technology to launch a fast PCR purification product (RapidTipTM) that enables single-step PCR cleanup in less than a minute simply by aspirating the PCR product into a functional pipette tip and dispensing purified DNA reaction solution. The work in the present proposal is to develop particles with surfaces that selectively bind the intercalating dyes used by Sage Science and to make the particles easily dispersible in water to facilitate rapid mixing and collection of impurities. We will also ascertain whether the size-selected DNA obtained with the Sage Science instrument and Diffinity purification is suitable for next generation sequencing library preparation. PUBLIC HEALTH RELEVANCE: Next generation sequencing of DNA is growing rapidly since it provides genetic information important to medical research and diagnosis. Size selection of DNA using gel electrophoresis is part of next generation sequencing protocols and a new product from Sage Science is streamlining that process. We propose a companion product that will reduce the time, cost and material waste associated with purifying the size-selected DNA obtained from the Sage Science process.