Digital PCR Technology and Workflow Overview

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Researchers performing sensitive applications such as copy number variation and rare target detection often find themselves struggling to identify faint genetic signals against an overwhelming background, sometimes as little as a single positive in a pool of negatives. It is indeed like finding the needle in a haystack. By discretizing the pool in a large number of individual reactions, digital PCR makes the single positive surprisingly easy to detect. It is in such instances where the lower limit of detection excludes conventional qPCR as a viable solution.

Digital PCR is a high-throughput, absolute quantitative PCR method that provides a sensitive and reproducible way of measuring the amount of DNA or RNA present in a sample. This method is similar to qPCR in the reaction assembly components and amplification reaction, but differs in the way the sample target is measured.
Principle
Comparison with droplet digital PCR (ddPCR)
Features and benefits of QIAGEN dPCR
Advantages over conventional qPCR
Applications
Principle
For QIAGEN’s nanoplate-based dPCR, one reaction mix per well is partitioned into a large number of individual partitions prior to the amplification step, resulting in one or very few templates being present in each partition. The partitions are small reaction compartments on prefabricated plates. Compartmented PCR reaction is cycled either in the presence of intercalating fluorescent dyes or labeled probes. Following PCR amplification, the number of positive vs. negative reactions is determined by the fluorescence intensity of each partition, imaging of which is done using a camera that gives a clear digital answer (yes/no). The absolute quantification of target is calculated using Poisson statistics.

Comparison with droplet digital PCR (ddPCR)
QIAGEN’s dPCR platforms are developed on nanoplate-based technology, offering significant benefits over droplet digital PCR (ddPCR). These include:

Fixed partitions prevent variation in size and coalescence
Sealed nanoplates prevent well to well contamination
• Faster readout possible due to simultaneous reading of all partitions of a sample
User-friendly, familiar plates are easy to pipet just like for qPCR 
Plates are amenable to front-end automation 

Features and benefits of QIAGEN dPCR
Fully integrated and rapid systems: Partitioning, thermocycling, imaging and analysis are all integrated into a single fully automated instrument. The novel digital PCR nanoplates deliver a streamlined workflow familiar to genomic researchers, with rapid imaging capabilities that enable results in under two hours, vs. more than four hours for current digital PCR systems. Standard plate formats allow front-end automation of sample preparation, which further reduces hands-on time.

Scalable instruments with high multiplexing capabilities: QIAGEN is developing scalable instrument formats designed to offer laboratories the highest degree of flexibility while ensuring full assay compatibility. The new systems will enable higher target multiplexing to increase the amount of information that can be obtained from a sample, thereby exceeding the capabilities of existing digital PCR platforms and matching those of qPCR systems.

Distinct plate configurations: Customers will be able to target varied applications with distinct plate types designed to accommodate a wide range of throughput and dynamic range requirements of applications from gene expression analysis to rare mutation detection, and can be used on all of the QIAGEN systems. Our nanoplate formats are currently in development.

Advantages over conventional qPCR
Absolute quantification
Provides an absolute count of target DNA copies per sample without the need for standard curves, reducing time and cost.

Highest precision and sensitivity
Partitions the sample into many small chambers on the dPCR nanoplate, resulting in enriched template concentration in the partitions.

1. Detect rare events (mutation) and low abundance targets (liquid biopsy) with increased sensitivity
2. Resolve very small differences in copy numbers and provide the precision required for CNV analysis

Elimination of PCR efficiency bias
Reduces error rates by removing the reliance on amplification efficiency of qPCR. Can maintain accuracy even in the presence of inhibitors.

Applications
It is highly suited for:
  
Liquid biopsy
Rare mutation detection
Copy number variation
Gene editing detection
Gene expression analysis 
Pathogen detection
NGS library quantification
Single-cell analysis
Microbiome analysis
GMO analysis
 
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Images
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Single well of a standard digital PCR plate
Single well of a standard digital PCR plate, where sample is divided into many separate partitions.
WF_0261_dPCR_Workflow
Simplified digital PCR workflow
Partitioning, thermocycling, imaging and analysis are all integrated into a single fully automated instrument, delivering results in under two hours, vs. more than four hours for current digital PCR systems. Plate formats also allow front-end automation of sample preparation, which further reduces hands-on time.
ILLU_1353_dPCR_Well_Anatomy
Plate-based digital PCR
In digital PCR (dPCR), the sample is divided into many separate partitions, with each partition containing either a single template molecule or none. Varied applications can be performed with distinct plate types designed to accommodate a wide range of throughput and dynamic range requirements, and can be used on all of the QIAGEN systems.