Complex Mechanisms of Mutation and Mutation Avoidance in Living Cells

活细胞突变和突变避免的复杂机制

• 批准号: 10581066
• 负责人: Eric Alan Josephs
• 金额: $ 8.27万
• 依托单位: UNIVERSITY OF NORTH CAROLINA GREENSBORO
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-17 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10581066
• 关键词: Administrative Supplement; Award; Biology; Cells; Complex; Containment; Detection; Emulsions; Encapsulated; Equipment; Exhibits; Faculty; Grant; Individual; Joints; Laboratories; Manuals; Methods; Mutation; North Carolina; Nucleic Acids; Oils; Postdoctoral Fellow; Preparation; Prevalence; Reference Standards; Reproducibility; Research; Sampling; Schools; System; Techniques; Technology; Time; Universities; Viral; Water; detection sensitivity; digital; equipment acquisition; improved; inhibitor; instrument; mutant; nanoengineering; nanoscience

We are requesting an administrative supplement to purchase equipment for R35 award 5R35GM133483 “Complex Mechanisms of Mutation and Mutation Avoidance in Living Cells” (project period: 09/17/2019 - 07/31/2024). We are requesting to purchase a Bio-Rad Automated Droplet Generator AutoDG Instrument (Bio- Rad item #1864101) for use with a QX200 Droplet Digital PCR (ddPCR) System and an associated Bio-Rad PX1 PCR Plate Sealer (Bio-Rad item #1814000) in order to significantly enhance the throughput and reproducibility of sample processing for the QX200 ddPCR system currently housed at the Joint School of Nanoscience and Nanoengineering (JSNN) of North Carolina A&T State University and UNC Greensboro (UNCG), where the PI is faculty (at UNCG and JSNN). ddPCR is an advanced PCR technique where individual nucleic acids from a sample are encapsulated in small oil-water emulsions prior to amplification by PCR, so that after the PCR cycles the emulsions that originally contained individual nucleic acids can be counted using fluorescent detection. ddPCR is at this point an established nucleic acid quantification technology that leads in precision and accuracy. Compared to traditional quantitative PCR (qPCR) methods such as the standard Applied Biosystems 7500 Real-Time PCR instrument (also at JSNN), ddPCR allows for absolute quantification of specific nucleic acids in a sample without the need for or variability of standard reference curves; provides increased sensitivity for the detection of rare mutants by over an order of magnitude (from >5% prevalence using qPCR to <0.1% prevalence); and exhibits less sensitivity to PCR inhibitors. These attributes will be crucial for emerging applications in projects related to the R35 grant. ddPPR and these requested equipment are expected to significantly help to advance and enhance several parallel lines of research associated with the R35 award in my laboratory. The reason for this is that the limitation of our ddPCR system is in its sample preparation—while the QX200 can perform nucleic acid quantification of 96 samples at a time, the standard equipment performs droplet preparation for only 8 samples at a time and this must be performed manually. The AutoDG system automates the simultaneous preparation of 96 samples, allowing us to maximize usage of this instrument and associated consumables. This automated processing will be necessary for the R35 research to significantly improve sample preparation throughput for the QX200 ddPCR system by orders of magnitude while reducing intra- and inter-user variability in preparation, all of which will be necessary to the completion of the research in a timely manner, to the highest quality standards, and at the sensitivity necessary to resolve key differences between experimental conditions. The PX1 PCR plate sealer will be necessary to maintain biosafety level- appropriate containment of Biosafety level 2 (BSL2) samples prior to and after droplet preparation.

我们正在请求行政补助以购买 R35 奖项 5R35GM133483 的设备 “活细胞突变与突变避免的复杂机制”（项目期间：2019年9月17日- 07/31/2024）我们请求购买一台 Bio-Rad 自动液滴发生器 AutoDG 仪器（Bio-Rad）。 Rad 商品 #1864101)，与 QX200 微滴式数字 PCR (ddPCR) 系统和相关 Bio-Rad 配合使用 PX1 PCR 封板机（Bio-Rad 货号 #1814000）可显着提高通量和 目前位于联合学院的 QX200 ddPCR 系统样品处理的重现性 北卡罗来纳 A&T 州立大学和北卡罗来纳大学格林斯伯勒分校纳米科学与纳米工程 (JSNN) (UNCG)，其中 PI 是教员（UNCG 和 JSNN），ddPCR 是一种先进的 PCR 技术，由个人负责。 在 PCR 扩增之前，将样品中的核酸封装在小的油水乳液中，以便 PCR 循环后，最初含有单个核酸的乳液可以使用以下方法进行计数 ddPCR 是目前领先的成熟核酸定量技术。 与标准应用等传统定量 PCR (qPCR) 方法相比。 Biosystems 7500 实时 PCR 仪（也在 JSNN），ddPCR 允许对特定物质进行绝对定量 样品中的核酸无需标准参考曲线或具有可变性即可提供增强的核酸； 检测罕见突变体的灵敏度超过一个数量级（使用 qPCR 从 >5% 的流行率到 <0.1% 的患病率）；并且对 PCR 抑制剂的敏感性较低，这些特性对于新兴技术至关重要。 与 R35 赠款相关的项目申请和这些请求的设备预计将得到满足。 极大地帮助推进和加强与 R35 奖项相关的几项平行研究 我的实验室的原因是我们的 ddPCR 系统的局限性在于其样品制备。 QX200一次可对96个样本进行核酸定量，标准设备执行 AutoDG 系统一次只能制备 8 个样品，且必须手动执行。 自动同时制备 96 个样品，使我们能够最大限度地利用该仪器， 这种自动化处理对于 R35 研究来说是必要的。 将 QX200 ddPCR 系统的样品制备通量提高几个数量级，同时减少 用户内部和用户之间的准备工作存在差异，所有这些对于完成研究都是必要的 及时、符合最高质量标准并具有解决关键差异所需的敏感性 PX1 PCR 板密封剂对于维持生物安全水平是必要的。 在液滴制备之前和之后适当容纳生物安全 2 级 (BSL2) 样品。