Absolute Quantification of Nucleic Acids with Droplet Digital PCR

Polymerase chain reaction (PCR) revolutionized molecular biology by enabling the exponential amplification of specific DNA sequences, with applications spanning from disease diagnosis to forensic analysis. Over the years, researchers rapidly evolved PCR technology, developing quantitative PCR (qPCR), which provides real-time target quantification via fluorescence monitoring. However, qPCR relies on standard curves, limiting its ability to detect rare variants or low-abundance targets in complex samples.

In this Innovation Spotlight, Marwan A. Alsarraj, global segment manager at Bio-Rad Laboratories, discusses how digital PCR eliminates the need for standard curves while achieving high sensitivity and precision in absolute quantification. He highlights how this strategy enables rare variant detection, including single-nucleotide mutations. Furthermore, Alsarraj explains the mechanism by which Droplet Digital™ PCR (ddPCR™) uses water-oil emulsion technology for absolute nucleic acid quantification through extensive sample partitioning and discusses why this technology has become essential for advancing cell and gene therapies.

What is digital PCR?

Digital PCR (dPCR) is a nucleic acid quantification technology that enables highly sensitive and precise measurement of targets. First conceptualized in the early 1990s, dPCR combines sample partitioning, end-point PCR, and data analysis to quantify target molecules without reliance on standard curves. Following PCR amplification, fluorescence readout scores partitions as positive if they contain amplified target product or negative if they do not. Researchers then use the proportion of positive partitions to calculate the absolute target concentration using Poisson statistics. By physically separating target molecules, dPCR minimizes competition between sequences and improves sensitivity relative to quantitative PCR, enabling robust detection of rare variants, including single-nucleotide mutations, within a high background of wild-type sequences.

What are the key advantages and challenges of digital PCR in research applications?

Digital PCR offers several distinct advantages for research applications, driven primarily by its sample partitioning approach and Poisson-based analysis. By dividing reactions into thousands of independent partitions, digital PCR achieves higher precision and sensitivity than conventional PCR and qPCR, making it well suited for detecting low-abundance targets and resolving subtle quantitative differences between samples. Absolute quantification without the need for standard curves further enhances reproducibility and cross-study comparability.

Historically, cost has been an important consideration among researchers with qPCR being the more economical option; however, this gap continues to narrow as dPCR platforms mature. In fact, a simpler multiplex assay design, improved tolerance to inhibitors, and reduced need for repeat measurements can make dPCR more cost-effective when sample material is limited or targets are rare. Concerns about workflow complexity and limited multiplexing or dynamic range have also diminished, as modern systems now offer streamlined, qPCR-like workflows, intuitive data analysis software, and expanded multicolor detection capabilities.

How does ddPCR technology work?

Droplet Digital PCR is a digital PCR method that achieves absolute nucleic acid quantification through extensive sample partitioning using water-oil emulsion droplet technology. During a ddPCR reaction, microfluidics and proprietary surfactant chemistries partition a sample containing the target nucleic acid into thousands of nanoliter-sized droplets, typically around 20,000. Each droplet then functions as an independent PCR microreactor, similar to a miniature test tube, and ideally contains zero, or at least one, target molecule.

Following droplet generation, researchers carry out PCR amplification simultaneously across all droplets using standard PCR probe-based chemistries, such as TaqMan assays. After amplification, the system individually assesses droplets for fluorescence to determine whether amplification occurred, classifying droplets as positive, indicating target amplification, or negative, indicating absence of target molecule. Researchers then use the fraction of positive droplets to calculate the concentration of the target sequence using Poisson statistics. This approach enables highly sensitive and absolute target quantification with minimal sample input. It is highly resistant to contaminants and reproducibility errors as it does not rely on standard curves, making ddPCR technology particularly well suited for detecting low-abundance targets and performing multi-sample type analyses, all while preserving precious samples.

What sets ddPCR technology apart from other digital PCR solutions, and what has the response been from researchers using this technology?

What makes ddPCR technology stand out from other dPCR approaches is the scale, consistency, and validation of its partitioning technology, as well as its long-standing adoption by the research community. For more than a decade, ddPCR has employed a patented water-oil emulsion system that reproducibly generates thousands of uniform nanoliter-sized droplets per sample, enabling highly reliable absolute quantification. This extensive partitioning supports the technology’s precision and accuracy, which have been independently evaluated using DNA reference standards by Australia’s National Measurement Institute and other international metrology organizations.¹ Such validation positions ddPCR technology as a leader for quantitative molecular measurements.

Researchers have responded to ddPCR technology with broad uptake across diverse fields, reflected in more than 12,000 peer-reviewed publications. Users frequently cite its robustness, reproducibility, accuracy, sensitivity, and ease of integration into existing PCR workflows. The ddPCR process itself is intentionally straightforward, where samples are partitioned into droplets, amplified using standard thermal cycling, and then analyzed to count positive and negative reactions. The system applies Poisson statistics to these counts to determine an absolute target concentration, providing researchers with high confidence quantitative data for a wide range of applications.

In what ways has ddPCR become essential for advancing cell and gene therapies, cancer research, and other research areas?

ddPCR technology has become essential in several key research areas because it supports multiple sample types, requires only small sample volumes, and delivers sensitive, absolute nucleic acid quantification. These features are particularly valuable when targets are present at low abundance or sample material is limited.

Specifically, in cell and gene therapy research, scientists successfully use ddPCR technology to assess critical quality attributes such as empty:full capsid ratio, vector copy number, viral titers, and residual host or plasmid DNA with a level of precision that does not depend on standard curves. This supports robust characterization of therapeutic constructs and strengthens confidence in manufacturing and quality control workflows, especially where regulatory expectations demand accurate, reproducible measurements.

In cancer research, researchers harness ddPCR’s high level of precision and sensitivity to detect rare mutations on wild-type backgrounds and low-abundance targets in liquid biopsy samples. This, in turn, allows researchers to monitor disease progression and treatment response with greater sensitivity than conventional PCR approaches.

Beyond these areas, ddPCR technology has become an essential tool for single-cell analysis, copy number variation, gene expression and miRNA studies, next-generation sequencing library analysis, and pathogen detection, particularly in applications where other tools struggle to deliver sufficient resolution or reproducibility.

Which recent technical innovations or emerging applications in ddPCR technology do you find most compelling?

Several recent innovations are making ddPCR solutions increasingly compelling in emerging applications, particularly in translational and cancer research settings. One of the most impactful is the move toward tumor-informed molecular residual disease monitoring using liquid biopsy samples, where ddPCR technology’s absolute quantification and tolerance to inhibitors enable sensitive and reproducible detection of low-frequency variants over time, such as circulating tumor DNA. This approach is well positioned to become routine as liquid biopsy workflows mature.

Advances in multiplexing, including six-color detection and beyond, also expand the scope of ddPCR assays. These capabilities allow researchers to assess multiple targets within a single well, reducing both sample requirements and overall cost while increasing the amount of data generated from each reaction.

Additionally, standardization and clinical readiness accelerate adoption. Ongoing efforts around International Organization for Standardization (ISO) and Clinical & Laboratory Standards Institute (CLSI) guidelines, along with the availability of in vitro diagnostic-ready platforms, improve assay consistency and are set to accelerate adoption into clinical settings. At the same time, harmonized preanalytical workflows for cell-free DNA are further enhancing reproducibility across laboratories. Together, these developments position ddPCR technology as the practical link between research and routine precision oncology care.