Digital PCR in a microplate format: high-throughput, precise DNA quantification

微孔板形式的数字 PCR：高通量、精确的 DNA 定量

• 批准号: 8780912
• 负责人: Kabir James Yamana
• 金额: $ 15.61万
• 依托单位: FORMULATRIX, INC.
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-08-01 至 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8780912
• 关键词: Affect; Automation; Behavior; Biological Assay; Complex; Copy Number Polymorphism; DNA; Data; Data Quality; Detection; Diagnostics Research; Fluorescent Dyes; Gene Expression; Genetic; Genomic DNA; Health; Housekeeping Gene; Image; Industry; Injection of therapeutic agent; Libraries; Liquid substance; Marketing; Measures; Microfluidics; Molds; Mus; Performance; Platelet Factor 4; Positioning Attribute; Preparation; Process; Production; Relative (related person); Reproduction; Research; Resolution; Respondent; Running; Sampling; Solutions; Specificity; Surveys; System; Technology; Testing; Time; cost; design; digital; instrumentation; meetings; new technology; next generation sequencing; performance tests; prototype; real world application; seal

DESCRIPTION (provided by applicant): Quantitative PCR (qPCR) is the current industry standard for quantification of DNA samples, and is used commonly in diagnostics and research. Digital PCR (dPCR) presents numerous advantages over qPCR such as increased precision and sensitivity and absolute quantification without comparison to standards. However, current digital PCR systems require complex workflows and are not easily scaleable. We have produced a prototype of an injection molded 96 sample microplate for digital PCR. Injection molding allows the production of the microplates with excellent feature reproduction and low costs per sample. Preliminary testing has shown the microplates to be capable of outperforming qPCR in several limited applications such as low copy number variation determination or low resolution sample quantitation. All 96 samples can be partitioned and thermocycled at once, which results in much higher throughput than any current dPCR system. Our system also features compatibility with liquid handlers and involves only a single liquid transfer step. However, our microplates are currently limited in application due to their modest number of partitions per sample (496), which is a rough measure of the resolution of a digital PCR system. We propose to develop a process for injection molding microplates with four times the number of partitions per sample (2000), dramatically increasing our precision, specificity, and dynamic range, and enabling the use of the microplates for a much wider range of applications such as library preparation for next-generation sequencing. We will produce an injection mold for mass production of digital PCR microplates, with multiple iterative steps of design and testing. In Aim 1, we will investigate the microfluidic performance of the microplates to determine the feasibility of the partition design and partitioning mechanism. In Aim 2, we will test the performance of PCR assays to determine the suitability of the microplates for PCR and thermal cycling. By completing the proposed research, we will have demonstrated the feasibility of our injection molded microplates for digital PCR, and developed a new system for high-throughput, low-cost absolute quantification in a familiar 96-well format. By giving users the precision of digital PCR with the simplicity, format, and throughput of quantitative PCR, we will transform digital PCR from a niche technology for precise quantification of a few samples to a serious competitor to quantitative PCR across a wide range of applications.

描述（由申请人提供）：定量 PCR (qPCR) 是当前 DNA 样本定量的行业标准，常用于诊断和研究。与 qPCR 相比，数字 PCR (dPCR) 具有许多优势，例如更高的精度和灵敏度以及无需与标准品进行比较的绝对定量。然而，当前的数字 PCR 系统需要复杂的工作流程并且不易扩展。我们生产了用于数字 PCR 的注塑 96 个样品微孔板原型。注塑成型可以生产具有出色特征再现性和每个样品低成本的微孔板。初步测试表明，微孔板在一些有限的应用中（例如低拷贝数变异测定或低分辨率样品定量）能够优于 qPCR。所有 96 个样品均可同时进行分区和热循环，这使得通量比任何当前的 dPCR 系统高得多。我们的系统还具有与液体处理机的兼容性，并且仅涉及单个液体转移步骤。然而，我们的微孔板目前在应用中受到限制，因为每个样品的分区数量有限 (496)，这是数字 PCR 系统分辨率的粗略测量。我们建议开发一种注射成型微孔板的工艺，每个样品的分区数量 (2000) 为四倍，从而显着提高我们的精度、特异性和动态范围，并使微孔板能够用于更广泛的应用，例如下一代测序的文库制备。我们将生产用于批量生产数字 PCR 微孔板的注塑模具，并包含多个设计和测试的迭代步骤。在目标 1 中，我们将研究微板的微流体性能以确定可行性 分区设计和分区机制。在目标 2 中，我们将测试 PCR 检测的性能，以确定微孔板是否适合 PCR 和热循环。通过完成拟议的研究，我们将展示用于数字 PCR 的注塑微孔板的可行性，并开发出一种以熟悉的 96 孔格式进行高通量、低成本绝对定量的新系统。通过为用户提供数字 PCR 的精度以及定量 PCR 的简单性、格式和通量，我们将把数字 PCR 从一种用于精确定量少数样品的利基技术转变为跨广泛应用的定量 PCR 的有力竞争对手。