Digital PCR Applications

Digital PCR is a specialized approach to nucleic acid detection and quantification that estimates absolute numbers of molecules through statistical methods. The digitization process distributes the PCR mix across thousands of microreactions so that each microreaction will effectively either contain one, zero, or just a handful of the target nucleic acid molecules. In effect, the original reaction is turned into many yes-or-no reactions. After counting the positive microreactions, simple statistics can be used to then determine the “absolute” quantity of the target molecule rather than a quantity estimated by comparing to a standard of known concentration.

As the name suggests, real-time PCR measures PCR amplification as it occurs. It focuses on the exponential phase because it provides the most precise and accurate data for quantitation. By comparing the Ct values of samples of unknown concentration with a series of standards, the amount of template DNA in an unknown reaction can be accurately determined.

Digital PCR works by digitizing a sample into many individual real-time PCR reactions; some portion of these microreactions contain the target molecule (positive) while others do not (negative). Following PCR analysis, the fraction of negative answers is used to generate an absolute answer for the exact number of target molecules in the sample, without the need for Ct values or reference to standards.

dPCR allows you to simply count the number of microchambers that have a specific or set of specific target molecules. This provides a mechanism for absolute quantification, which lets you determine the number of molecules in your sample without the need for standard curve as would be typical in a qPCR reaction. This standard curve–free absolute quantification is especially beneficial for laboratories in need of highly precise and consistent quantification methods. Since dPCR also allows you to identify handfuls of rare molecules in an overwhelming number of normal or background molecules, it is excellent for applications such as detecting rare oncogenic mutations in circulating free DNA. Multiplexing enables you to perform absolute quantification to several target molecules in parallel.

Additionally, dPCR takes an endpoint measurement in order to determine concentration and is overall less sensitive to some common inhibitors that can interfere with qPCR reaction kinetics and efficiency. This improved performance in the face of inhibitors is an important attribute for research labs working with environmental or precious samples.

dPCR is well suited to performing rare allele detection, measurement of copy number variation, viral titer measurement, quantification of next-generation sequencing libraries, and detection of rare targets from environmental samples such as wastewater. Specific applications available for absolute quantification include:

• Oncology research—improve detection of mutations and quantification of low allele fractions in challenging samples such as liquid biopsy with high specificity, precision, and accuracy. Applications include analysis of rare or limited samples, rare target quantification, gene expression, and target multiplexing.
• Cell and gene therapy research—absolute quantification of viral vector backbones commonly used in cell and gene therapy research such as adenoassociated virus (AAV) and cytomegalovirus (CMV). Applications include residual plasmid DNA, viral titer quantification, NGS library quantification, and plasmid or vector construct analysis.
• Environmental surveillance—accurately measure viral load and pathogens or other biomarkers in environmental samples such as wastewater by absolute quantification with improved performance compared to qPCR in the face of common PCR inhibitors. Applications include target multiplexing, infectious disease detection, and absolute quantification.
• Gene editing—detect genome editing events in a fast and reliable way, especially those created using nucleases that cause double-stranded breaks, such as CRISPR-Cas9. Applications include absolute quantification, target multiplexing, and analysis of rare events.
• Inherited disease research—accurate and precise quantification of rare mutations associated with human diseases along with exceptional discrimination of CNVs with high copy number. Applications include copy number variation (CNV), genotyping, and target multiplexing.