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.
Thesaurus Terms: Affect;Automation;Behavior;Biological Assay;Complex;Copy Number Polymorphism;Cost;Data;Data Quality;Design;Detection;Diagnostics Research;Digital;Dna;Fluorescent Dyes;Gene Expression;Genetic;Genomic Dna;Health;Housekeeping Gene;Image;Industry;Injection Of Therapeutic Agent;Instrumentation;Libraries;Liquid Substance;Marketing;Measures;Meetings;Microfluidics;Molds;Mus;New Technology;Next Generation Sequencing;Performance;Performance Tests;Platelet Factor 4;Positioning Attribute;Preparation;Process;Production;Prototype;Real World Application;Relative (Related Person);Reproduction;Research;Resolution;Respondent;Running;Sampling;Seal;Solutions;Specificity;Surveys;System;Technology;Testing;Time;
