Digital PCR infrastructure to enhance research and HQP training in biology

定量PCR （qPCR）彻底改变了生态学和进化、分子生物学、农业、医学和土木工程等许多领域，允许在一系列生物样品中快速定量核酸。然而，使用传统的qPCR对目标模板DNA进行量化依赖于标准稀释序列，其准确性随着目标丰度的降低而降低，实验之间的标准化可能具有挑战性，并且假阴性和假阳性的比例可能很高。对于我们实验室中需要检测和定量低拷贝数目标DNA模板的许多应用（例如稀有等位基因的检测，生物入侵的早期阶段，稀有保护物种或湖泊沉积物中的古代eDNA），数字PCR （dPCR）提供了一个重要的技术飞跃，允许直接计数单个目标模板。在女王大学生物系，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的局限性影响了研究进展和HQP培训，影响了我们进行新颖研究的能力，也影响了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，我们整个系31名专任教师、120名研究生和60名本科生只有一台任何类型的qPCR机。为了纠正这些问题，我们建议购买一台高精度BioRad QX200液滴数字PCR工作站，该工作站将放置在我们共享的部门分子核心实验室中，并提供给我们部门的所有人，优先考虑本提案的共同申请人。

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.