Digital PCR (dPCR): Principle, Workflow, Types, Applications & Examples

Table of Contents

• Introduction to Digital PCR
• Objectives of Digital PCR (dPCR)
• Requirement for Digital PCR (dPCR)
• Principle of Digital PCR (dPCR)
• Steps of Digital PCR (dPCR)
• Types of Digital PCR (dPCR)
• Examples of Digital PCR (dPCR)
• Applications of Digital PCR (dPCR)
• Advantages of Digital PCR (dPCR)
• Limitations of Digital PCR (dPCR)
• Conclusion
• References

Introduction to Digital PCR

• Digital PCR (dPCR) was invented in 1999 by Kenneth W. Kinzler and Bert Vogelstein. It is a molecular technique in which DNA samples are diluted and distributed into microtiter plates or many small reaction compartments.
• The method uses fluorescent probes to detect the presence of specific DNA sequences. These probes emit signals when the target DNA segment is amplified, enabling precise detection and quantification of DNA molecules.
• Digital PCR estimates the number of DNA molecules statistically by counting positive and negative reactions across many microreactions.
• Unlike traditional Polymerase Chain Reaction, which measures the amount of accumulated amplification product after each cycle, digital PCR focuses on counting the fraction of negative microreactions.
• By determining how many reactions do not contain the target DNA, dPCR can calculate the absolute number of DNA copies present in the sample using statistical analysis.
• Compared with conventional PCR approaches, digital PCR provides more accurate quantitative results because it counts individual DNA molecules rather than relying on amplification curves.
• Digital PCR operates with minimally diluted samples, allowing highly sensitive detection even when the target DNA sequence is extremely rare within a large population of wild-type sequences.
• Because of this high sensitivity, dPCR is widely used for detecting rare mutations, particularly in studies analyzing tumor heterogeneity in oncology.
• The technique also plays an important role in liquid biopsy applications, where circulating tumor DNA in blood can be detected and quantified.
• Additionally, digital PCR is used in prenatal diagnosis to identify and quantify inherited genetic mutations with high precision.

Objectives of Digital PCR (dPCR)

• The main objective of digital PCR (dPCR) is to enable precise detection and highly sensitive quantification of nucleic acids from minimally diluted DNA samples containing the target DNA segment.
• This is achieved by partitioning a single Polymerase Chain Reaction reaction into numerous small subreactions, allowing each partition to function as an independent amplification chamber.
• Each of these subreactions undergoes amplification under optimal PCR conditions, ensuring that the target nucleic acid, if present, can be efficiently amplified.
• When a subreaction produces amplification, it indicates that the target nucleic acid molecule is present in that specific partition.
• Conversely, when a subreaction does not show amplification (negative subreaction), it indicates the absence of the target nucleic acid in that partition.
• By analyzing the number of positive and negative partitions, digital PCR enables accurate counting of target molecules present in the original sample.
• Another important objective of digital PCR is to allow precise absolute quantification of nucleic acids without relying on an external reference or standard curve, which is commonly required in other PCR-based quantification methods.

Requirement for Digital PCR (dPCR)

The materials which are involved in dPCR include:

Conventional PCR Components

Digital PCR (dPCR) requires the basic components used in conventional Polymerase Chain Reaction, including primers, deoxynucleotide triphosphates (dNTPs), DNA polymerase enzyme, reaction buffers, and essential ions, which are necessary for the amplification of DNA.

Reporter Molecules

• A reporter molecule is required for detecting amplification during the reaction. This reporter may be either a fluorescent dye or a probe that generates a detectable signal when amplification of the target DNA occurs.
• Fluorescent DNA-binding dyes are commonly used because they bind to double-stranded DNA and enable detection of amplified products.
• Hydrolysis probes may also be used for more specific detection of the target DNA sequence.

Thermocycler

A thermocycler is required to perform the repeated heating and cooling cycles necessary for DNA amplification, including denaturation, primer annealing, and extension steps.

Partition Chambers

• Partition chambers are required to divide the PCR reaction mixture into a large number of microchambers or microreactions, which allows individual amplification events to occur independently.
• Different platforms can be used to create these partitions, including microfluidic chips, microarrays, spinning microfluidic discs, and droplet-based systems generated through oil–water emulsions.

Fluorescence Analyzer

• A fluorescence analyzer is used to detect and quantify amplified DNA in each partition by measuring the fluorescent signal produced during amplification.
• Since a single subreaction may contain more than one target molecule, the fluorescence data obtained are analyzed using the Poisson distribution, which allows accurate estimation of the absolute number of target DNA molecules in the original sample.

Principle of Digital PCR (dPCR)

• Digital PCR (dPCR) is a highly precise and sensitive molecular technique used for detecting rare genetic events such as single-nucleotide mutations within a population of wild-type DNA sequences.
• In this method, the competition between DNA targets in the reaction mixture is reduced through partitioning, which helps overcome the inherent difficulty of amplifying rare sequences present in very small quantities.
• By minimizing this competition among DNA templates, digital PCR enables highly sensitive detection and accurate absolute quantification of nucleic acids.
• Because of this sensitivity and precision, digital PCR is widely applied in fields such as oncology for detecting rare mutations, as well as for the detection and quantification of viruses and pathogens, and in single-cell analysis studies.
• The experimental procedure begins by diluting the DNA sample to be analyzed and evenly partitioning it into microtiter plates, typically distributing the templates so that approximately one DNA template molecule is present per two wells.
• After partitioning, PCR amplification is carried out under optimal reaction conditions, allowing the target DNA segment to replicate and produce multiple copies of the sequence of interest.
• The amplified DNA fragments, known as amplicons, are then hybridized with a reporter molecule, typically a fluorescent DNA-binding probe.
• These fluorescent probes enable sequence-specific detection of amplified products, and different fluorophores can be used to identify specific DNA sequences.
• Following amplification and probe binding, the fluorescent signals generated in each reaction partition are detected and analyzed using statistical approaches.
• A critical step in digital PCR is the division of the reaction mixture into numerous microwells or partitions before amplification begins.
• Each partition may contain zero, one, or more template DNA molecules, depending on the dilution and distribution of the sample.
• The efficiency of amplification can vary slightly from sample to sample, but the effects of minor inhibitors or reduced accessibility of the target DNA have less influence on the results due to the partitioned nature of the reaction.
• After amplification, the resulting amplicons are detected using fluorescent probes, which reveal whether the target DNA is present in each partition.
• Partitions that emit fluorescence are considered positive reactions (represented as 1), indicating the presence of the target DNA molecule.
• Partitions that do not produce fluorescence are considered negative reactions (represented as 0), indicating the absence of the target DNA molecule in that partition.
• The ratio of positive to negative partitions forms the basis for quantifying the number of target molecules in the sample.
• By combining sample partitioning with statistical analysis using the Poisson distribution, digital PCR provides more precise and reliable quantification compared to traditional Polymerase Chain Reaction methods.

Steps of Digital PCR (dPCR)

Based on the principle of digital PCR, the procedure can be summarized into five main steps: sample preparation, partitioning, amplification, hybridization, and analysis with quantification.

1. Sample

• The sample contains the specimen carrying the target nucleic acid sequence, which may include DNA, RNA, or complementary DNA (cDNA) obtained from different biological sources.
• Along with the specimen, the reaction mixture also contains the standard components required for conventional Polymerase Chain Reaction, including primers, deoxynucleotide triphosphates (dNTPs), DNA polymerase enzyme, buffers, ions, and other reaction components necessary for amplification.
• A reporter molecule is also included in the mixture, which may be a hydrolysis probe or a fluorescent DNA-binding dye, enabling detection of amplified DNA.
• The biological specimen used in digital PCR can originate from different sources such as tissue samples, body fluids, or other biological materials containing the nucleic acids of interest.

2. Partitioning

• After the reaction mixture is thoroughly prepared, the sample is partitioned into numerous microchambers or wells so that amplification can occur independently in each compartment.
• Typically, the total reaction volume is around 20 μL, which is then distributed into multiple wells, commonly a 96-well microchamber plate.
• Partitioning is performed such that on average one template DNA molecule is distributed across two wells, ensuring statistical separation of DNA templates.
• During this step, the target DNA molecules are randomly distributed among thousands or even millions of small partitions.
• Two major types of partitioning approaches are used in digital PCR:
• Water-in-oil droplet emulsification:
• This technique is commonly known as droplet digital PCR (ddPCR).
• The reaction mixture is divided into thousands to millions of droplets ranging from picoliters (pL) to nanoliters (nL) in size.
• These monodisperse droplets are generated rapidly using microfluidic chips, which apply forces that break the aqueous phase into droplets within an oil phase.
• During thermal cycling, droplets may coalesce due to temperature fluctuations, so surfactants are used to stabilize the water-in-oil emulsions.
• Droplet digital PCR provides greater scalability and improved cost-effectiveness compared with microchamber-based systems.
• Microchamber-based partitioning:
• This was the first digital PCR format to be developed.
• In this method, the reaction mixture is distributed into thousands of microscopic wells embedded within a solid chip.
• Compared with droplet systems, microchamber dPCR offers higher reproducibility and easier automation because it does not require precise droplet emulsification.
• However, this approach is limited to a fixed number of partitions and generally involves higher operational costs.
• With advances in technology, modern digital PCR instruments can rapidly partition samples into hundreds, thousands, or even millions of subreactions, providing different levels of detection resolution.

3. Amplification

• After partitioning, each microreaction is subjected to thermal cycling conditions to perform amplification using a thermocycler.
• Each partition acts as an independent PCR reaction chamber, where wells containing the target DNA sequence will produce positive amplification reactions.
• The standard PCR amplification cycle consists of three major steps:
• Denaturation:
• The DNA is heated to approximately 95 °C, causing the hydrogen bonds between complementary base pairs to break, resulting in the separation of the double-stranded DNA into single strands.
• Annealing:
• The temperature is then lowered to approximately 55 °C to 72 °C, allowing primers to bind to their complementary sequences on the template DNA.
• This step prepares the template for subsequent synthesis and elongation of the DNA strand.
• Extension:
• During this step, the temperature is raised to around 75 °C to 80 °C, which is optimal for DNA polymerase activity, enabling elongation of the DNA strand and synthesis of new DNA molecules.
• The efficiency of amplification can vary between samples, especially if minor inhibitors are present or if target DNA accessibility is reduced.
• Delayed amplification due to accessibility issues generally does not significantly affect quantitative results, unlike in other PCR techniques.
• As long as positive reactions can be clearly distinguished from negative ones, general inhibition does not significantly affect result interpretation.

4. Hybridization

• After the amplification process is completed, the amplified DNA products are hybridized with fluorescent probes.
• These probes enable the differentiation between positive and negative subreactions based on fluorescence signals.
• Because each amplified product originates from a single DNA template molecule, the resulting PCR products are homogeneous in sequence, making fluorescence-based detection technologies highly suitable for digital PCR analysis.
• Molecular beacons are currently widely used for detection in digital PCR reactions due to their high specificity.
• In addition to molecular beacons, hydrolysis probes can also be used to detect and differentiate amplified products.
• In some cases, positive reactions may fail to produce fluorescence, which may occur due to delayed amplification caused by primer accessibility problems or point mutations located at the probe-annealing site.

5. Analysis and Quantification

• Fluorescent probes enable visual detection of positive and negative subreactions, but due to the random distribution of template molecules, not every positive partition necessarily contains only one target molecule.
• When the sample concentration exceeds a certain level or the number of partitions is limited, some partitions may contain multiple template molecules.
• To correct for this and determine the absolute quantity of target molecules, a statistical correction based on the Poisson distribution is applied.
• Quantification is achieved by counting the number of positive and negative partitions.
• The ratio of positive reactions (p) to the total number of partitions is used to calculate the average number of target molecules per partition (λ) using the Poisson equation.
• The calculated value is then multiplied by the number of partitions per microliter to estimate the total number of target molecules in the sample.
• Negative reactions play a particularly important role in digital PCR quantification, as they help establish the ratio of positive to negative reactions and provide a basis for accurate molecule counting.
• The Poisson formula used for this statistical modelling is:

Copies per partition (λ) = −ln(1 − p)

• In this equation, p represents the fraction of positive reactions, and the calculated λ value represents the average number of target molecules present in each partition.
• The combination of sample digitization and Poisson statistical analysis enables digital PCR to produce highly precise and reliable quantitative results.

Types of Digital PCR (dPCR)

Digital PCR can be divided into two main types based on the partitioning technique used in the reaction process. These include:

Droplet Digital PCR (ddPCR)

• In droplet digital PCR (ddPCR), the reaction mixture is dispersed into an immiscible oil phase, forming thousands to millions of tiny droplets.
• Each droplet functions as an individual microreactor where amplification of the target nucleic acid occurs independently.
• The droplets typically range in size from picoliters to nanoliters, allowing large-scale partitioning and highly sensitive detection of nucleic acid molecules.

Microchamber Digital PCR

• In microchamber digital PCR, the reaction mixture is partitioned into numerous small chambers or wells.
• These microscopic wells are embedded within a solid chip, allowing the reaction mixture to be separated into thousands of independent microreactions.
• Each chamber acts as a separate PCR reaction compartment, where amplification of the target DNA sequence occurs if the template molecule is present.
• Despite differences in the partitioning method used in droplet-based or microchamber-based systems, the fundamental principle and amplification process of Polymerase Chain Reaction remain unchanged in digital PCR.

Examples of Digital PCR (dPCR)

1. QX700™ E System – Bio-Rad Laboratories

• This system uses Droplet Digital PCR (ddPCR) technology based on water–emulsion droplet partitioning.
• In this method, the sample is fractionated into up to 17,000 droplets, where each droplet functions as an independent PCR microreaction.
• The droplets are then subjected to amplification by providing optimal thermal cycling conditions.
• Each droplet containing the target nucleic acid sequence produces a positive amplification signal, which is used to calculate precise and absolute quantification of the target molecule.
• Up to 5 μL of sample can be partitioned into 192 wells, and the final results are typically obtained within approximately 5 hours.
• The system utilizes water-emulsion technology combined with microfluidics to achieve high-precision partitioning and amplification.
• Other ddPCR instruments developed by Bio-Rad include:
• QX Continuum System
• QX700 S System
• QX700 HT System
• QX600 System
• QX200 System
• QX ONE System
• QXDx AutoDG System

2. QuantStudio Absolute Q Digital PCR System – Thermo Fisher Scientific

• This instrument is a plate-based digital PCR system that utilizes Microfluidic Array Plate (MAP) technology.
• MAP technology integrates several steps of digital PCR including compartmentalization, thermal cycling, and data acquisition within a single instrument platform.
• Each MAP plate contains 16 separate digital PCR reaction units.
• Every reaction unit is composed of 20,480 fixed microchambers, enabling high-resolution partitioning of the PCR mixture.
• These microchambers are connected to a distribution flow system that supplies PCR reagents to each chamber.
• After compartmentalization, PCR amplification occurs inside each microchamber, and chambers showing successful amplification are counted.
• This system is capable of analyzing more than 95% of the input sample, whereas many other digital PCR instruments typically analyze only 25–60% of the sample.

3. Nio® Digital PCR System – Stilla Technologies

• The Nio® Digital PCR system is an all-in-one instrument designed for user-friendly operation and is available in three different configurations.
• The system is fast, highly automated, and integrates advanced hardware and software components.
• This technology is also known as Crystal Digital PCR.
• In this system, samples are partitioned into thousands of droplet crystals, forming a large array of individual reaction compartments.
• Each droplet crystal functions as a separate microreaction chamber where amplification takes place independently.
• After amplification, targets are labeled with fluorophores and detected through fluorescence analysis.
• The system can analyze up to seven fluorescence channels simultaneously, enabling multiplex assays capable of quantifying up to 21 different targets at the same time.

4. QIAcuityDx Digital PCR System – Qiagen

• This system is a nanoplate-based digital PCR platform designed for in vitro diagnostic applications.
• It uses automated multiplex digital PCR technology for accurate nucleic acid quantification.
• The nanoplate technology integrates partitioning, thermocycling, and fluorescence image analysis into a fully automated workflow.
• Depending on the nanoplate format, up to 96 samples can be analyzed on a single plate.
• For diagnostic applications, the QIAcuityDx Nanoplate 26K is commonly used, which contains 24 wells with thousands of nanopartitions.
• These wells include a continuous loading system, reducing manual handling and making the workflow minimal hands-on.
• The QIAcuityDx Control Software manages the entire integrated system, including:
• robotic gripper for nanoplate handling
• partitioning module
• thermocycler
• fluorescence imaging module
• The system can process up to four plates in a single run, requiring approximately 120 minutes for the first plate and around 80 minutes for each subsequent plate.
  

Applications of Digital PCR (dPCR)

• Digital PCR (dPCR) has become a valuable molecular technique with applications across various fields due to its high sensitivity, precision, and ability to perform absolute quantification of nucleic acids.
• One major application of digital PCR is viral pathogen detection. Because dPCR requires only a small amount of sample, it is particularly useful for detecting viruses where specimen availability is often limited, making traditional detection methods more challenging.
• Digital PCR is also widely used in high-resolution gene expression analysis. In this method, quantification of nucleic acids is calculated using the Poisson distribution, which ensures that the numerical results obtained are independent of other samples.
• Because of this statistical independence, when the results are compared with reference genes, the analysis produces more statistically significant and reliable gene expression data.
• Another important application of digital PCR is the detection of copy number variation (CNV). The technique allows the precise detection and quantification of small differences in gene copy numbers, even when the variation occurs in very small percentages, providing a much higher level of precision than many conventional methods.
• Digital PCR is also useful in single-cell analysis. When studying individual cells, an initial amplification step is usually required before further downstream molecular analysis.
• However, digital PCR can produce highly accurate results even when the target nucleic acid levels are very low, meaning that fewer preamplification steps are required compared with traditional approaches, improving accuracy in single-cell experiments.
• Another important use of digital PCR is the detection of rare mutation abundance. The partitioning of samples into numerous microreactions and the enrichment effect produced by this partitioning allow high-resolution detection of somatic mutations present within disease-affected samples.
• In addition to these major uses, digital PCR is also applied in environmental pathogen detection, where it helps identify microorganisms present in environmental samples.
• The technique is further used in library quantification for Next‑Generation Sequencing, ensuring accurate preparation of sequencing libraries.
• Digital PCR is also applied in genetically modified organism (GMO) detection and food testing, where it enables accurate identification and quantification of specific genetic modifications present in food products.

Advantages of Digital PCR (dPCR)

• Digital PCR (dPCR) offers several advantages over traditional methods such as Polymerase Chain Reaction due to its high sensitivity, accuracy, and ability to provide absolute quantification of nucleic acids. 
• The partitioning of the sample into thousands or millions of microreactions ensures more precise and high-resolution results, as each partition functions as an independent amplification reaction.
• Since digital PCR relies on the Poisson distribution for determining the absolute copy number of target molecules, there is no need to depend on external references or standard curves for quantification.
• The presence of inhibitors in the sample has minimal impact on the amplification process, because the reaction is distributed across multiple independent partitions.
• Digital PCR is highly effective for detecting rare mutations, allowing the analysis of complex mixtures containing very small amounts of mutant DNA within a large background of normal DNA.
• The method is capable of detecting even very small percentage changes in nucleic acid quantity, and these changes are reported linearly in relation to the number of DNA copies present.
• Digital PCR is therefore considered a calibration-free, highly sensitive technique that enables accurate absolute quantification of nucleic acids.

Limitations of Digital PCR (dPCR)

• Digital PCR (dPCR), despite its advantages, also has several limitations that may affect its practical application in molecular analysis. 
• Partitioning of the sample into thousands or millions of microreactions requires high precision and careful handling, which can make the process time-consuming and technically demanding.
• The overall cost of performing digital PCR is relatively high because it requires specialized reagents, fluorescent probes, and advanced instrumentation, increasing operational expenses.
• In droplet digital PCR (ddPCR) systems, precise emulsification of the sample into stable droplets is essential, and maintaining droplet stability throughout thermal cycling can make the procedure more complex and time-consuming.
• Microchamber-based digital PCR systems are limited by a fixed number of partitions, which can restrict the level of resolution achievable, and these systems often involve higher costs compared with other PCR methods.
• During quantification, careful statistical analysis is required when applying the Poisson distribution, because calculation errors may lead to inaccurate estimation of the absolute number of target molecules. 

Conclusion

Digital PCR (dPCR) is considered a third-generation PCR technique that enables highly accurate detection and quantification of nucleic acids even when only small amounts of sample are available. 

The partitioning of samples into numerous small microchambers or microreactions plays a key role in its effectiveness, as this separation allows high-resolution detection and reduces inaccuracies during quantification.

Because of its exceptional sensitivity and precise quantification capability, digital PCR is particularly valuable for the detection of rare genetic mutations, making it highly useful in fields such as oncology and molecular diagnostics.

Although the technique has certain limitations, including higher costs and longer processing times, ongoing technological developments aim to overcome the practical and technical challenges associated with digital PCR.

Overall, digital PCR represents a significant advancement in nucleic acid analysis compared with traditional methods like Polymerase Chain Reaction, offering greater sensitivity, precision, and reliability in molecular research and diagnostics. 

References

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