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

描述(申请人提供)：定量聚合酶链式反应(QPCR)是目前DNA样本定量的行业标准，广泛用于诊断和研究。数字聚合酶链式反应(DPCR)与定量聚合酶链式反应(QPCR)相比具有许多优点，如更高的精密度和灵敏度，以及绝对定量而不与标准相比较。然而，目前的数字聚合酶链式反应系统需要复杂的工作流程，并且不易扩展。我们已经制作了一个用于数字聚合酶链式反应的注射成型96个样品微孔板的原型。注塑成型使微型板的生产具有出色的特征重现性和较低的每件样品成本。初步测试表明，在一些有限的应用中，如低拷贝数变异测定或低分辨率样品定量，微孔板的性能优于qPCR。所有96个样本都可以一次被分割和热循环，这导致了比目前任何dPCR系统都要高得多的吞吐量。我们的系统还具有与液体处理机兼容的特点，并且只涉及单一的液体转移步骤。然而，我们的微孔板目前的应用受到限制，因为它们的每个样本的分隔数不多(496个)，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板的工艺，每个样品的隔板数量是(2000)的四倍，极大地提高了我们的精密度、特异性和动态范围，并使微板能够用于更广泛的应用，如为下一代测序准备文库。我们将生产一种用于批量生产数字PCR微孔板的注射模具，包括多个迭代的设计和测试步骤。在目标1中，我们将研究微板的微流控性能，以确定其可行性 分区设计和分区机制。在目标2中，我们将测试聚合酶链式反应检测的性能，以确定微孔板在聚合酶链式反应和热循环中的适用性。通过完成拟议的研究，我们将证明我们的注射成型微板用于数字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聚合酶链式反应的精确度和定量聚合酶链式反应的简单性、格式和吞吐量，我们将把数字聚合酶链式反应从一种用于精确量化少数样本的利基技术转变为定量聚合酶链式反应在广泛应用中的有力竞争对手。

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.