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Short answer: ddPCR (droplet digital PCR) splits one PCR reaction into thousands of nanolitre-scale droplets, amplifies each droplet separately, and counts how many end up fluorescence-positive. The fraction of positive droplets, corrected with Poisson statistics, gives an absolute copy number without a standard curve.
Definition and scope
Droplet digital PCR is one format of digital PCR (dPCR), in which the sample is partitioned so that each partition holds zero, one or a few target molecules. In ddPCR the partitions are droplets suspended in oil. The result is a concentration of target molecules, not a quantification cycle (Cq) that has to be converted through a calibration curve. For the broader choice between the two approaches, see the digital PCR vs qPCR comparison; this page covers what ddPCR is, why the arithmetic works, and what a report has to contain.
How ddPCR works in practice
The description below follows the system published by Hindson and colleagues (Analytical Chemistry, 2011), which is the paper that introduced the droplet-based platform. Figures are given as that paper reports them for that system; they are not specifications for any instrument sold today.
- Partitioning. In the 2011 system, about 20,000 monodisperse droplets of 1 nL each were formed from a 20 µL reaction. Applying a vacuum to the outlet wells drew sample and oil through a flow-focusing junction.
- End-point reading. After thermal cycling, each droplet is read for fluorescence. In the authors’ words, a simple threshold on fluorescence amplitude assigns each droplet as positive or negative.
- Counting and correction. The positive fraction is converted to a concentration with a Poisson correction, which the authors describe as extending the dynamic range into the region where droplets hold, on average, more than one copy.
- No calibrators. The authors report quantitation without external calibrators or endogenous controls, because the count itself carries the information that a standard curve supplies in qPCR.
Why the Poisson correction matters: a worked example
If copies were counted simply as positive droplets, any droplet that started with two or more copies would be undercounted. The correction uses the negative droplets instead. With a mean of λ copies per droplet, the fraction of empty droplets is e−λ, so λ = −ln(fraction negative). The numbers below are hypothetical counts chosen for the arithmetic, using the 2011 system’s partition sizes (20,000 droplets of 1 nL, 20 µL reaction).
| Step | Value |
|---|---|
| Droplets accepted | 20,000 |
| Positive / negative droplets | 5,000 / 15,000 |
| Fraction negative | 15,000 / 20,000 = 0.75 |
| λ = −ln(0.75) | 0.288 copies per droplet |
| Copies in the 20,000 nL reaction | 0.288 × 20,000 ≈ 5,754 |
| Uncorrected count (positives only) | 5,000 |
The gap between 5,754 and 5,000 is the multiple-copy droplets that a plain count misses. It widens as the positive fraction rises, which is why the correction, and the assumptions behind it, belong in the report.
Related concepts
ddPCR sits next to several concepts that are easy to conflate:
- qPCR reads fluorescence every cycle and depends on a calibration curve or reference for absolute numbers; see qPCR and RT-qPCR and standard-curve building.
- Digital PCR is the general category; droplets are one way to make the partitions.
- Multiplexing places more than one assay in the same reaction; see multiplexed PCR.
- Copy number is a common application: the 2011 paper used the platform for DNA copy number, and the topic is covered in copy number of a gene.
Where it matters for labs: what to report
The dMIQE Group’s 2020 update (Huggett et al., Clinical Chemistry 66(8):1012) sets out the minimum information for publishing digital PCR experiments. These are the items from it that most directly follow from the ddPCR arithmetic above:
| Report this | What dMIQE 2020 says |
|---|---|
| Partition number | The average number and range of measured partitions is necessary. |
| Partition volume | Partition volumes, whether assumed or directly measured, and the software versions used should be reported. |
| Threshold | Reports should include the method of manual threshold setting. |
| Negative controls | Reactions containing the reaction mix without template are required, plus a control with the same DNA/RNA background but without the targeted sequence. |
| Low-level false positives | The false-positive frequency defines the limit of detection or quantification. |
| Items that do not apply | The revised checklist adds a comments section for the rationale. |
The same guideline lists the conditions that must hold for results to follow the Poisson distribution: clear separation of positive and negative partitions, equal partition volumes, and random distribution of the target. If a run fails one of these, the corrected concentration is not trustworthy, so check them before reading the number. For the equivalent checklist on the qPCR side, see MIQE guidelines; for the statistics, see the Poisson distribution.
Common misunderstandings
- “Absolute” means error-free. It means the concentration comes from counting and Poisson statistics rather than a calibration curve. It still depends on the partition volume being right and on the threshold separating positives from negatives, which is why dMIQE asks for both.
- The positive fraction is the copy number. Only when almost every positive droplet holds one copy. The worked example shows the difference.
- Sensitivity figures transfer between systems. The 0.001% mutant fraction (BRAF V600E) that Hindson et al. reported, which they put at 1,000 times lower than real-time PCR, is a result for their 2011 system and assay, not a property of every ddPCR run.
- Negative controls are optional with counting methods. dMIQE 2020 calls for them, because false-positive droplets set the practical limit of detection.
FAQ
What does ddPCR stand for?
Droplet digital PCR: a digital PCR format in which the reaction is divided into droplets suspended in oil that are amplified and read individually.
Does ddPCR need a standard curve?
No. Hindson et al. report quantitation without external calibrators or endogenous controls, because the Poisson-corrected count of positive and negative droplets gives the concentration directly.
How many droplets does a reaction produce?
In the 2011 system described by Hindson et al., about 20,000 droplets of 1 nL from a 20 µL reaction. Report the actual number for your instrument; dMIQE 2020 asks for the average number and range of measured partitions.
What should a ddPCR methods section include?
At minimum the items in the table above: partition number and range, partition volume and software version, the threshold method, and the no-template control and the background control without the target.
Sources
- Hindson BJ et al. High-throughput droplet digital PCR system for absolute quantitation of DNA copy number. Analytical Chemistry 2011. DOI 10.1021/ac202028g, PMID 22035192. pmc.ncbi.nlm.nih.gov/articles/PMC3216358 (checked 2026-10-29).
- Huggett JF; dMIQE Group. The Digital MIQE Guidelines Update: Minimum Information for Publication of Quantitative Digital PCR Experiments for 2020. Clinical Chemistry 2020;66(8):1012. DOI 10.1093/clinchem/hvaa125, PMID 32746458. academic.oup.com/clinchem/article/66/8/1012/5880067 (checked 2026-10-29).
