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.

我们正在申请行政补充，以购买R35奖5 R35 GM 133483的设备 “活细胞中突变和避免突变的复杂机制”（项目期间：09/17/2019 - 2024年7月31日）。我们正在申请购买Bio-Rad自动液滴发生器AutoDG仪器（Bio-Rad Automated Droplet Generator AutoDG Instrument）。 Rad产品编号1864101），用于QX 200微滴数字PCR（ddPCR）系统和相关Bio-Rad PX 1 PCR板密封器（Bio-Rad产品编号1814000），以显著提高通量， 目前在联合学校的QX 200 ddPCR系统的样品处理的再现性 北卡罗来纳州A&T州立大学和格林斯伯勒的纳米科学和纳米工程（JSNN） （UNCG），其中PI是教师（在UNCG和JSNN）。ddPCR是一种先进的PCR技术， 在通过PCR扩增之前，将来自样品的核酸包封在小的油-水乳液中，使得 在PCR循环之后，可以使用 荧光检测ddPCR在这一点上是一种已建立的核酸定量技术， 精确度和准确度。与传统的定量PCR（qPCR）方法相比， Biosystems 7500实时PCR仪器（也在JSNN），ddPCR允许绝对定量特异性 不需要标准参考曲线或标准参考曲线的可变性;提供增加的 检测罕见突变体的灵敏度超过一个数量级（从使用qPCR的>5%患病率到使用qPCR的>5%患病率）。 <0.1%患病率）;并且对PCR抑制剂表现出较低的敏感性。这些属性对于新兴市场至关重要。 与R35赠款有关的项目的申请。ddPPR和这些要求的设备预计将 显著有助于推进和加强与R35奖相关的几个平行研究领域， 我的实验室原因是我们的ddPCR系统的局限性在于其样本制备-而 QX 200一次可对96个样本进行核酸定量，标准设备 一次仅制备8个样品的液滴，并且这必须手动进行。AutoDG系统 自动化同时制备96个样品，使我们能够最大限度地利用该仪器， 相关耗材。这种自动化处理对于R35研究是必要的， 将QX 200 ddPCR系统的样品制备通量提高几个数量级， 用户内部和用户之间的差异，所有这些都是完成研究所必需的， 及时地、以最高的质量标准、以解决关键分歧所需的敏感度 实验条件之间。PX 1 PCR平板灭菌将是维持生物安全水平所必需的- 在液滴制备之前和之后，对生物安全2级（BSL 2）样品进行适当的密封。