Digital PCR infrastructure to enhance research and HQP training in biology

定量聚合酶链式反应(QPCR)使生态学和进化论、分子生物学、农业、医学和土木工程等领域发生了革命性的变化，使一系列生物样本中的核酸得以快速定量。然而，使用传统的qPCR定量靶模板DNA依赖于标准稀释系列，其中准确性随着靶丰度的降低而下降，实验之间的标准化可能是具有挑战性的，并且可能存在高的假阴性和阳性率。对于我们实验室中需要检测和量化低拷贝数目标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、稀有物种保护关注的物种或湖泊沉积物中的古代EDNA)，数字聚合酶链式反应(DPCR)提供了一项重要的技术飞跃，允许直接计数单个目标模板。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，传统的定量聚合酶链式反应的局限性正在影响研究进展和HQP培训，影响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)抛开传统定量聚合酶链式反应的局限性，我们只有一台超额认购的任何类型的定量聚合酶链式反应机器，供我们整个系的31名全职教师、120名研究生和60名本科生使用。为了纠正这些问题，我们建议购买一台高精度的BioRad QX200 Droplet Digital PCR工作站，该工作站将安装在我们部门共享的分子核心实验室中，并向我们部门内的所有人提供，优先考虑这项提议的共同申请者。

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.