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）彻底改变了生态和进化，分子生物学，农业，医学和土木工程中的许多领域，从而可以在一系列生物样品中快速定量核酸。然而，使用传统QPCR对目标模板DNA进行量化依赖于标准稀释系列，在该系列中，准确性会随着目标丰度较低而降低，实验之间的标准化可能是具有挑战性的，并且可能存在很高的假否定性和积极因素。对于我们的实验室中的许多应用，需要检测和量化低拷贝数目标DNA模板（例如检测稀有等位基因，生物入侵的早期阶段，稀有物种的保护问题或湖泊沉积物中的古代Edna），数字PCR（DPCR），提供一个重要的技术飞跃向前，允许直接计数单个目标模板。在皇后大学生物学系中，我们面临两个挑战：1）对于需要高度敏感，定量DNA检测的许多项目，传统QPCR的局限性损害了研究进度和HQP培训，我们进行新颖研究和进行新颖研究的能力，以及我们在最高质量期刊上发布的能力。 2）无论传统QPCR的局限性如何，我们的整个31名全职教职员工，> 120名研究生和> 60名本科论文专业的学生，​​只有一台任何QPCR机器的任何类型的QPCR机器。为了纠正这些问题，我们建议购买高精度的Biorad QX200液滴数字PCR工作站，该工作站将安置在我们的共享部门分子核心实验室中，并可以在我们的部门内使用，并优先在此提案中向共同申请者提供。