Digital PCR (dPCR) is a refinement of conventional PCR that provides absolute quantification of nucleic acids without needing a standard curve. The sample is partitioned into thousands of tiny individual reactions, and after amplification, the number of positive versus negative partitions is counted.
How Digital PCR Works
- Partitioning
The PCR mixture containing the sample, primers, probe, and polymerase is divided into thousands of nanoliter-sized partitions. This can be done using a chip with micro-wells, droplets in an oil emulsion (droplet digital PCR), or other methods. Each partition contains either zero or at least one copy of the target DNA.
- Amplification
The partitioned sample undergoes standard thermal cycling. In each partition, the PCR proceeds independently. Partitions containing target DNA produce amplified product, while empty partitions produce none.
- End-Point Detection
After amplification, each partition is analyzed for fluorescence. Partitions containing amplified target DNA are scored as positive (fluorescent), while those without are scored as negative. No quantification cycle (Ct) is needed—it is simply a yes-or-no readout per partition.
- Poisson Statistics
Since the partitioning is random, some partitions may contain multiple copies of the target. Poisson statistics are used to calculate the absolute number of target molecules in the original sample based on the proportion of negative partitions.
- Applications
Digital PCR is highly precise and is used for quantifying rare mutations, detecting low-abundance pathogens, analyzing copy number variations, and verifying NGS results. It is less sensitive to PCR inhibitors than qPCR and provides absolute quantification without standards.
Practical dPCR Workflow
Prepare the PCR master mix containing the sample DNA (1–100 ng), primers, probe, and droplet-generation oil. For droplet digital PCR (ddPCR), load the mixture into a droplet generator that partitions the sample into ~20,000 uniform nanoliter droplets in a water-in-oil emulsion. For chip-based dPCR, the sample is loaded into a microfluidic chip with prefabricated microwells (typically ~20,000 wells). Transfer the partitioned sample to a thermal cycler and amplify to end-point using standard cycling conditions. After amplification, read the fluorescence of each partition using a dedicated reader. Partitions with target DNA show fluorescence above a manually set threshold and are scored as positive. Apply Poisson statistics: λ = −ln(1 − p) where p is the fraction of positive partitions; the absolute target concentration (copies/µL) is λ divided by the partition volume. Use at least 10,000 accepted partitions for reliable quantification. Include a no-template control and a positive control with known copy number. For rare mutation detection, use dual fluorescent probes — a wild-type probe labeled with FAM and a mutant probe with HEX — to distinguish mutant from wild-type molecules in the same reaction.
Real-World Application
In liquid biopsy for cancer monitoring, ddPCR detects circulating tumor DNA (ctDNA) mutations such as EGFR T790M in non-small cell lung cancer. Plasma DNA (10 ng) is extracted from blood and analyzed with mutation-specific probes. ddPCR resolves mutant allele frequencies as low as 0.01%, far below the detection limit of Sanger sequencing. The absolute copy number of mutant molecules per mL of plasma is calculated, enabling longitudinal tracking of tumor burden and early detection of treatment resistance.
