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

从尺寸选定的 DNA 中快速去除嵌入剂以进行下一代测序

• 批准号: 8314446
• 负责人: LEWIS J ROTHBERG
• 金额: $ 9.96万
• 依托单位: DIFFINITY GENOMICS, INC.
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-08-15 至 2013-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8314446
• 关键词: Address; Adopted; Adsorption; Agitation; Aspirate substance; Binding; Biochemistry; Buffers; Caring; Chemistry; Collection; Companions; DNA; DNA Sequence; DNA-Directed DNA Polymerase; Diagnosis; Diagnostic; Dyes; Electrophoresis; Excision; Fluorescence; Gel; Genetic; Genomics; Homidium Bromide; Individual; Intercalating Agents; Left; Libraries; Licensing; Link; Location; Marketing; Measures; Medical; Medical Research; Medicine; Metric; Nucleic Acids; One-Step dentin bonding system; Oranges; Polymerase Chain Reaction; Preparation; Procedures; Process; Protocols documentation; Reaction; Sampling; Savings; Science; Sepharose; Silicon Dioxide; Slice; Solutions; Speed; Surface; System; Technology; Therapeutic; Time; Triazines; Tube; Universities; Validation; Water; Work; base; cost; drug development; ds-DNA; gel electrophoresis; improved; infrared spectroscopy; instrument; instrumentation; next generation; novel; particle; silochrome; success; translational study; trend; wasting; water solubility

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.

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“个性化医疗”的基石，其中治疗课程与个人的基因组成相关联。从电泳后的凝胶中提取DNA以获得大小选择的DNA片段是所有主要下一代测序平台协议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，该产品可以用电“切换”凝胶中所需的DNA片段，从而避免了物理提取DNA的需要。由于其速度和节省劳动力的协议，Sage Science Pippin Prep正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，以去除用于识别条带的插层染料，因为它们会干扰下一代测序文库制备的后续过程。Diffinity Genomics提出开发一种基于功能移液管尖端的单步纯化过程，该过程将在不到60秒的时间内消除插入染料，同时保留所需的DNA。