Digital PCR Defined: Absolute Quantification Benefits and Purposes

Digital PCR is decades old, but recent advances have led to a renaissance of this method. With high sensitivity, precision, and throughput, and no calibration or standard curves required, scientists increasingly apply digital PCR to measure the concentration of target sequences across a range of research fields. In this article, learn about digital PCR and how it works, its advantages, and some key applications of this method.

What Is Digital PCR?

Digital PCR, or dPCR, is a molecular biology technique that detects and quantifies nucleic acid targets. By partitioning a sample into many individual reactions, scientists can measure whether a target is present or absent in each reaction, providing an absolute target concentration within the sample.

Cancer researchers Bert Vogelstein and Kenneth Kinzler invented digital PCR in the early 1990s, and they later demonstrated proof-of-concept for this method by using it to detect mutant oncogenes in stool samples from patients with colorectal cancer.¹

How does digital PCR work?

To perform digital PCR, researchers partition or divide a DNA sample via serial dilution. This results in thousands to millions of microreactions containing either zero, one, or a very small number of DNA molecules that are amplified individually.² Researchers add a fluorescent probe into the reaction, which hybridizes to the target sequence and yields a digital distribution of DNA targets, detected as the presence or absence of fluorescence in each microreaction. By counting all the positives and using Poisson correction, scientists can calculate the absolute quantity of the target in the overall sample.

Digital PCR vs qPCR

Researchers often use quantitative PCR (qPCR) and dPCR for similar applications, but they have differences in their quantification method. qPCR measures the fluorescence signal emitted during PCR cycles in real time. Fluorescence intensity relates to DNA concentration, where the more intense the fluorescence, the higher the concentration of DNA in the sample.³ This technique uses a standard curve or established reference sample to relatively quantify the target.

In contrast, dPCR measures the presence or absence of fluorescence in each microreaction and has a lower limit of detection, allowing scientists to identify a single positive target, such as a rare allele or mutation, in a large sample of more abundant DNA species.⁴ Unlike qPCR, digital PCR does not require a standard curve or reference sample.⁴

Advantages of digital PCR

There are several advantages of digital PCR, including its high sensitivity and precision, reproducibility, and rapidity.^2,3 Because dPCR uses such small reaction volumes, the target concentration is increased while the “noise” of non-target fragments is reduced. This means researchers can accurately quantify rare targets in complex samples, making dPCR highly applicable in a range of research areas. Digital PCR is also less affected by the presence of PCR inhibitors than other PCR methods.³

With recent technological advances such as droplet-based microfluidics, digital PCR is now relatively low-cost and easy-to-use, resulting in an increase in its popularity in the last decade.⁵ Some researchers are currently developing portable digital PCR devices, with a 2024 study describing a smartphone-operated digital PCR device for point-of-care testing in multiple fields.⁴

Digital PCR Protocol: Reagents, Instruments, Assay, and Analysis

Digital PCR reagents

Digital PCR requires similar reagents to other PCR methods, and researchers often purchase these as ready-made kits. Typical digital PCR reagents include primers, fluorescent probes, and a PCR master mix containing Taq polymerase, deoxynucleotide triphosphates (dNTPs), reaction buffer, and magnesium chloride. For droplet digital PCR (ddPCR), a droplet generation oil or cartridge is also used.

Digital PCR instruments

There are several commercial digital PCR instruments available, depending on the preferred approach. They are typically comprised of a partition generator, thermal cycler, and a fluorescence reader or image processing system.⁴

Digital PCR assay

1. 1. Sample partitioning

Scientists typically perform sample partitioning using either a nanoplate or droplet-based approach. The former method, known as chip-based digital PCR (cdPCR), partitions the sample into microchambers on a solid chip. The latter is droplet digital PCR (ddPCR), which uses droplet-based microfluidics to partition the sample.⁶ ddPCR is currently more cost-effective and scalable, while cdPCR is more reproducible and highly automated.²

2. 2. Signal amplification

Once the sample has been partitioned, researchers use a thermal cycler to simultaneously amplify the target in each microchamber or droplet.²

3. 3. Signal detection

Once the digital PCR microreactions have been amplified, researchers use the fluorescence reader or image processing system to detect a positive or negative fluorescence readout from each microreaction.⁷

4. 4. Signal quantification and digital PCR analysis

The final step in a basic digital PCR assay is to assess the proportion of fluorescence-positive to fluorescence-negative reaction chambers. As the concentration of the target increases, so does the chance of co-occupancy of multiple DNA targets in each microreaction; the number of positives is Poisson-corrected to provide an accurate calculation of DNA concentration in the sample.²

Digital PCR Applications

Digital PCR’s high sensitivity has led scientists to apply this method across a range of research fields and clinical applications. Its original application in detecting rare oncogene mutations is still relevant today. Clinicians often use digital PCR to perform non-invasive liquid biopsy, detecting relevant nucleic acid markers derived from cell-free DNA and RNA, circulating tumor cells, extracellular vesicles, and exosomes.⁸ This allows clinicians to diagnose cancer, predict and monitor treatment outcomes, and provide personalized medicines. Researchers have also recently used multiplexed droplet digital PCR to simultaneously detect eight different tumor types based on their unique DNA methylation profile.⁹

Digital PCR is also highly relevant in the field of infectious disease research. A consensus among virologists is that digital PCR consistently outperforms qPCR across all basic parameters, making it ideal for detecting and quantifying mutations and viral copy number.⁵ Researchers have used digital PCR in critical care medicine settings to detect bacterial and fungal infections in blood, and viruses such as SARS-CoV-2.¹⁰

Clinicians can also use digital PCR in non-invasive prenatal testing (NIPT) as a simple alternative to the more complex and expensive next-generation sequencing that is commonly used.¹¹ The presence of cell-free fetal DNA in maternal blood samples means that scientists can use digital PCR to detect genetic mutations, copy number variations, and chromosomal abnormalities, such as trisomy.¹¹