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.

我们要求使用行政补充来购买R35奖5R35GM133483的设备 “活细胞中突变和突变的复杂机制”（项目期：09/17/2019-- 07/31/2024）。我们要求购买Bio-Rad自动液滴发电机Autodg仪器（Bio-- RAD项目＃1864101）与QX200液滴数字PCR（DDPCR）系统和相关的Bio-Rad一起使用 PX1 PCR板密封剂（Bio-Rad项目＃1814000），以显着增强吞吐量和 QX200 DDPCR系统的样品处理的可重复性目前位于联合学校 北卡罗来纳州A＆T州立大学和UNC Greensboro的纳米科学和纳米工程（JSNN） （UNCG），PI是教师（在UNCG和JSNN）。 DDPCR是一种高级PCR技术 来自样品的核酸在通过PCR放大之前将小的油水乳液封装在小型油水乳液中，以便 PCR循环后，可以使用最初包含单个核酸的乳液来计数 荧光检测。 DDPCR目前是一项已建立的核酸定量技术，导致 精度和准确性。与传统的定量PCR（QPCR）方法（例如应用标准）相比 生物系统7500实时PCR仪器（也在JSNN），DDPCR可以绝对量化特定 样品中的核酸无需或标准参考曲线的变异性；提供增加 通过超过一个数量级来检测稀有突变体的敏感性（使用qpcr> 5％的患病率> 5％ <0.1％患病率）；并且对PCR抑制剂的敏感性较小。这些属性对于新兴至关重要 与R35赠款有关的项目中的申请。 DDPPR和这些要求的设备预计将 大大有助于促进和增强与R35奖相关的几条平行研究。 我的实验室。这样做的原因是我们的DDPCR系统的限制是在其样本准备中 QX200可以一次执行96个样本的核酸定量，标准设备执行 一次只需准备8个样品，必须手动执行此样品。 AutoDG系统 自动化96个样本的简单准备，使我们能够最大化该乐器的使用情况和 关联的消耗品。 R35研究需要进行自动处理才能显着 通过降低的数量级来改善QX200 DDPCR系统的样品制备吞吐量 准备中的内部和用户间变异性，所有这些都是必要的 及时的方式，达到最高质量标准，并以解决关键差异所需的灵敏度 在实验条件之间。 PX1 PCR板密封剂必须保持生物安全水平 - 在液滴制备之前和之后，适当遏制生物安全水平2（BSL2）样品。