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.
