Digital PCR infrastructure to enhance research and HQP training in biology

数字 PCR 基础设施可加强生物学研究和 HQP 培训

• 批准号: RTI-2021-00120
• 负责人: Lougheed, Stephen
• 金额: $ 9.25万
• 依托单位: Queen's University
• 依托单位国家: 加拿大
• 项目类别: Research Tools and Instruments
• 财政年份: 2020
• 资助国家: 加拿大
• 起止时间: 2020-01-01 至 2021-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=718198
• 关键词: Digital; PCR; infrastructure; enhance; research

Quantitative PCR (qPCR) revolutionized many fields within ecology and evolution, molecular biology, agriculture, medicine, and civil engineering, allowing for rapid quantification of nucleic acids in a range of biological samples. Yet quantification of a target template DNA using traditional qPCR relies on a standard dilution series, where accuracy decreases with lower target abundance, standardization among experiments can be challenging, and there can be high rates of false negatives and positives. For many applications in our laboratories requiring detection and quantification of low copy number target DNA template (e.g. detections of rare alleles, early stages of biological invasions, rare species of conservation concern, or ancient eDNA in lake sediments), digital PCR (dPCR) provides an important technological leap forward, allowing for direct counts of individual target templates. In the Department of Biology at Queen's University, we face two challenges: 1) For many projects that need highly-sensitive, quantitative DNA detection, the limitations of traditional qPCR are compromising research progress and HQP training, our ability to undertake novel research, and our ability to publish in the highest-quality journals. 2) Irrespective of the limitations of traditional qPCR, we have only one over-subscribed qPCR machine of any kind for our entire department of 31 full-time faculty, >120 graduate students, and >60 undergraduate thesis students. To rectify these issues, we propose to purchase a high precision BioRad QX200 Droplet Digital PCR workstation that will be housed in our shared departmental molecular core lab, and made available to all within our department, with priority given to co-applicants on this proposal.

定量PCR（qPCR）彻底改变了生态学和进化、分子生物学、农业、医学和土木工程中的许多领域，允许快速定量一系列生物样品中的核酸。然而，使用传统qPCR对靶模板DNA的定量依赖于标准稀释系列，其中准确度随着较低的靶丰度而降低，实验之间的标准化可能具有挑战性，并且可能存在高的假阴性和假阳性率。对于我们实验室中需要检测和定量低拷贝数靶DNA模板的许多应用（例如检测稀有等位基因、生物入侵的早期阶段、受保护的稀有物种或湖泊沉积物中的古eDNA），数字PCR（dPCR）提供了重要的技术飞跃，允许直接计数单个靶模板。在皇后大学生物系，我们面临着两个挑战：1）对于许多需要高灵敏度定量DNA检测的项目，传统qPCR的局限性正在影响研究进展和HQP培训，我们进行新研究的能力，以及我们在最高质量期刊上发表文章的能力。2)不考虑传统qPCR的局限性，我们只有一台超额订购的qPCR机器，用于我们整个部门的31名全职教师，>120名研究生和>60名本科论文学生。为了纠正这些问题，我们建议购买一个高精度的BioRad QX200 Droplet Digital PCR工作站，该工作站将安装在我们共享的部门分子核心实验室中，并提供给我们部门内的所有人，并优先考虑共同申请人。