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被广泛用于翻译研究，并有望成为“个性化医学”的基石，在这种医学中，治疗过程与个人的基因构成有关。在所有主要的下一代测序平台的协议中，从凝胶中提取DNA以获得大小选择的DNA片段是文库准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，从而消除了物理提取DNA的需要。由于其速度和省力协议，Sage Science Pippin Prep正在迅速被快速增长的下一代测序市场采用。不幸的是，对于Pippin Prep，仍然需要进行相对劳动密集型的纯化，以去除用于识别条带的插层染料，因为它们干扰了下一代测序文库准备的后续过程。迪菲尼蒂基因组公司建议开发一种基于功能吸管尖端的一步纯化工艺，该工艺将在不到60秒的时间内消除插层染料，同时保留所需的DNA。 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。颗粒表面经过特殊配置，可以选择性地吸附或排斥各种溶液成分，从而可以在去除不需要的杂质的同时，将所需的成分保留在溶液中。迪菲尼蒂利用了这种差异 利用吸附技术推出一种快速的聚合酶链式反应纯化产品(RapidTipTM)，只需将聚合酶链式反应产物吸入功能移液管尖端并分配纯化的DNA反应溶液，即可在不到一分钟的时间内完成一步聚合酶链式反应的清除。本提案中的工作是开发具有表面选择性地结合所使用的插层染料的颗粒 Sage Science，并使颗粒易于分散在水中，便于快速混合和收集杂质。我们还将确定用圣贤科学仪器和差异纯化获得的大小选择的DNA是否适合于下一代测序文库的准备。 与公共卫生相关：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。使用凝胶电泳法选择DNA大小是下一代测序协议的一部分，Sage Science的一款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.