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

描述（由申请人提供）：定量PCR（qPCR）是目前用于定量DNA样本的行业标准，通常用于诊断和研究。数字PCR（dPCR）相对于qPCR具有许多优点，例如提高的精确度和灵敏度以及无需与标准品进行比较的绝对定量。然而，目前的数字PCR系统需要复杂的工作流程，并且不容易扩展。我们已经生产了一个注塑成型的96样品的数字PCR微孔板的原型。注塑成型允许生产具有优异特征再现和每个样品低成本的微孔板。初步测试表明，微孔板能够在几种有限的应用中优于qPCR，例如低拷贝数变异测定或低分辨率样品定量。所有96个样品可以一次分区和热循环，这导致比任何当前dPCR系统高得多的通量。我们的系统还具有与液体处理器的兼容性，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适中（496），这是数字PCR系统分辨率的粗略衡量标准。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是2000的四倍，大大提高了我们的精度，特异性和动态范围，并使微孔板能够用于更广泛的应用，如下一代测序的文库制备。我们将生产用于大规模生产数字PCR微孔板的注塑模具，其中包含多个迭代设计和测试步骤。在目标1中，我们将研究微板的微流控性能，以确定可行性 分区设计和分区机制。在目标2中，我们将检测PCR检测试剂盒的性能，以确定微孔板对PCR和热循环的适用性。通过完成拟议的研究，我们将证明我们的注塑微孔板用于数字PCR的可行性，并开发出一种新的系统，用于以熟悉的96孔格式进行高通量，低成本的绝对定量。通过为用户提供数字PCR的精确度以及定量PCR的简单性、格式和通量，我们将数字PCR从用于精确定量少数样品的利基技术转变为定量PCR在广泛应用中的有力竞争者。

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.