Fundamentals of digital PCR
What is digital PCR?
Digital PCR (dPCR) is a method for absolute quantification of nucleic acid concentrations through the combination of limiting dilution, end-point PCR and Poisson statistics.
In the digital PCR process, a digital PCR machine divides a PCR reaction made of template nucleic acids, primers, probes, nucleotides, enzymes and buffers into thousands of microreactions. Each partition effectively contains none, one or several of the target nucleic acid molecules. The digital PCR instrument amplifies the microreactions separately. At the end of amplification process, fluorescent probes are used to detect the presence or absence of products in each partition. Positive reactions with template nucleic acids would emit a fluorescent signal (on), negative reactions without the target nucleic acid would remain dark (off).
What makes digital PCR digital is precisely this on/off nature of the dPCR process. The term “digital assay” originates from digital computers, where computations are performed in logic circuits with information encoded in a series of ones and zeroes. The advantage of digital signals is that the instrument must only distinguish between two signals, rather than a full range of possibilities. This holds true when the principle is transferred to biological sensors. In digital PCR, the sensor must only distinguish between a positive and negative partition, which simplifies the instrumentation.
After analysing the number of positive and negative reactions, the “absolute” number of molecules present in the sample can be calculated using Poisson statistics. Unlike qPCR, dPCR does not rely on standard curves for nucleic acid quantification. The precise nature of digital PCR makes it well suited to detection of rare events in complex backgrounds.
Even when you know the answer to what is digital PCR, how can you choose between end-point PCR, qPCR or dPCR for your application? Find more information in the next section on PCR technologies comparison.
The principle of dPCR
Brief history of digital PCR
Digital PCR analysis has been around for more than three decades, but recent improvements of the digital PCR system and chemistry have let the technique truly flourish. What milestones lie in the history of turning PCR digital?
- 1988: The concept of digital PCR is first described
- 1999: The dPCR method is further developed; first study coining the term “digital PCR” is published
- 2006/2007: Release of first commercial dPCR system based on microfluidic chips and microarrays
- 2010: First commercial dPCR machine using spinning microfluidic discs is introduced
- 2011: Launch of first commercial dPCR instrument based on water-oil emulsion/droplets
- 2013: First commercial microplate-based dPCR machine is introduced
- 2020: Release of first commercial nanoplate-based dPCR system
How do these various digital PCR instruments compare to one another? Read in our section comparing digital PCR methods, including chip-based digital PCR, droplet digital PCR (ddPCR) and nanoplate dPCR.
How does digital PCR work?
Digital PCR (dPCR) is based on the principle of end-point PCR, but the PCR reaction mix is split into thousands of single partitions. The nucleic acid template is randomly distributed across all available separations and amplified individually. The amplification target is detected at the end of the PCR process by measuring the presence or absence of fluorescence of sequence-specific DNA probes or intercalating dyes, turning PCR digital.
As the PCR reaction is partitioned randomly, it is possible that a positive reaction contains more than one target molecule. To account for microreactions with more than one template sequence, Poisson statistics is applied.
Poisson statistics in digital PCR
In digital PCR analysis, the Poisson model is used to determine the probability of a microreaction receiving zero, one, two, three, four or five copies of the target molecule based on the following formula:
As the figure above shows, for a low concentration like λ = 0.1, most of the partitions will contain zero copies of the target molecule and nearly all positive partitions will contain only one copy of the target molecule. For medium concentrations like λ = 0.5, some positive partitions will likely contain more than one copy of the target molecule. For higher concentrations like λ = 5, most of the positive partitions will contain more than one copy of the target molecule and nearly no partition will contain zero copies of the target molecule.
The best way to illustrate how Poisson statistics works in digital PCR analysis is through an example.
You mix 5 µL DNA sample with 3 µL mastermix and 4 µL primers for a total dPCR reaction of 12 µL. After you run your digital PCR system, you quantify 4000 positive partitions out of 8000 valid partitions.
How to calculate copies/partition and copies/µL in digital PCR?
The total number of copies of the target molecule in all valid partitions of a well is calculated by multiplying the copies of the target molecule per partition with the number of valid partitions. Based on the known number of copies of the target molecule per partition (λ) and the partition volume, the copies per microliter can also be calculated.
The 3013 copies/μl is the concentration, which is the standard readout in the dPCR results. To calculate the copies of the target molecule in the reaction volume, you multiply by the input reaction volume. In case the input reaction volume is 12 μl, the copy number in the input reaction is 3013 x 12 = 36,156 copies.
With a reported concentration of 3013 copies/μl and 5 μl sample in 12 μl reaction volume, the 5 μl original sample contains (12/5) x 3013 = 7231 copies/μl. Therefore, the copies in the 5 μl sample are 5 x 7231 = 36,156 copies of target DNA, which is equal to the copies in the total reaction.
Digital PCR workflow in action
If you have familiarized yourself with what digital PCR is and how does digital PCR work, the next step is to set up your first digital PCR reaction. The nanoplate-based digital PCR workflow follows three basic steps:
1. Prepare and load your digital PCR instrument
- Measure DNA amount and purity
- Set up your digital PCR system by defining dPCR parameters, such as priming, cycling, imaging profiles, reaction mixes, samples, controls and plate layout
- Prepare your reaction mix in a pre-plate
- Pipette prepared reaction mix into nanoplate
- Seal with foil and manual rolling
- Hold the plate carefully at the side edges or transport by tray so that the reaction mix stays the bottom of the input well
- Load plate and start your digital PCR machine
2. Run and amplify
- The plate is processed in the priming/rolling module where the reaction mix of each well is partitioned into a thousand little individual reactions
- PCR is performed on the thermocycler
- Presence of template material within individual partitions leads to fluorescence signal that is detected during imagingThe images are processed in software suite of the digital PCR instrument.
3. Analyze dPCR results
- Depending on your dPCR system, select your application to view a heatmap, histogram, 1D scatterplot, 2D scatterplot
- Use option to change threshold setting and recalculate results based on the new settings
For more tips on how to set up your first digital PCR reaction, watch a webinar with tips and tricks for your dPCR workflow or read answers to top 10 FAQs on optimizing dPCR assays on the QIAcuity.
Digital PCR applications
What makes PCR digital is the ability to achieve absolute quantification of DNA, cDNA or RNA with a clear yes/no answer on the presence of target molecules. Because dPCR analysis occurs at the end of the amplification without reliance on standard curves, the technique is more precise and less susceptible to inhibitors, errors and amplification biases than other PCR methods.
Using a digital PCR machine makes the most sense when working on applications dealing with small quantities and rare targets from complex samples. Most digital PCR applications require exceptionally high sensitivity, specificity and discriminatory power. Such digital PCR applications include:
- Rare sequence detection
- Analysis of copy number variation (CNV)
- Quantification of rare mutations including in liquid biopsy testing
- Single-cell analysis
- Measurement of viral load
- Gene expression and miRNA analysis
- Pathogen detection
- Quantification of next-generation sequencing (NGS) libraries
- Detection of rare targets from environmental samples, including wastewater or sewage
Discover these and other nanoplate digital PCR applications in more detail in our digital PCR applications page.
Benefits and limitations of digital PCR
Just like with any technique, digital PCR analysis has particular benefits and limitations.
What are the advantages of using a digital PCR system?
- Absolute target quantification – No need for standards, reference curves or extrapolations
- High tolerance to inhibitors – Thanks to partitioning and endpoint measurement, dPCR efficiency remains unaffected by PCR inhibitors.
- Superior precision – Partitioning in digital PCR results in thousands of data points and more accurate results at the end of the amplification process, making digital PCR analysis suitable for detection of small fold change differences.
- Improved sensitivity – digital PCR systems offer an improved limit of detection (LOD) as a smaller reaction volume increases the effective concentration of the target nucleic acid. Partitioning also positively affects enrichment as the target is separated from interfering compounds. The overall ratio of target versus background is improved as wild-type sequences and high-copy templates are diluted in each partition. Hence, rare mutations and low-abundance targets are more accurately detected using dPCR than other PCR technologies.
- High reproducibility – digital PCR analysis remains reproducible across laboratories as amplification efficiency bias is drastically reduced.
- Cost-efficiency – Sample volumes and reagents are kept at minimal, lowering experimental costs; multiplexing possibilities enable higher throughput and productivity.
What are some digital PCR disadvantages?
- Dynamic range – The number of microunits in the digital PCR instrument is limited so the range of the number of DNA/RNA copies that can be detected by the digital PCR machine tends to be slightly narrower compared to qPCR.
- Large amplicons – dPCR is not suitable for analysis of very large amplicons
- Bias and variance – DNA denaturation during partitioning, where single strands separate into two different units, could cause overestimation; template linkage, sample inhomogeneity, partition volume variance and inhibiting factors, such as “molecular dropout” could cause underestimation.
Select publications using digital PCR
|Viral load monitoring
concentration in RNA
extracts from wastewater
to measure target
|GBA variant analysis
in patients with
or Lewy body dementia
|Detected copy number
variations using dPCR
of a multilocus
adaptive walk affecting
|NRAMP1 copy number
and expression analysis
|Development of single
of absolute lengths
and quantities of individual