dPCR

Digital PCR (dPCR) is a method for absolute quantification of nucleic acids that relies on partitioning a sample into many discrete reactions. End-point PCR is performed in each partition, and partitions are read as positive or negative for the target sequence. The fraction of positive partitions, analyzed with Poisson statistics, yields an absolute estimate of target molecules without the need for standard curves.

In practice, the sample is divided into thousands to millions of partitions. This can be achieved with droplets in oil in droplet digital PCR (ddPCR) or with isolated chambers on a microfluidic chip in chip-based dPCR. After amplification, partitions are scored by fluorescence to determine their status. The approach can be extended to RNA quantification by combining with reverse transcription (RT-dPCR).

Compared with quantitative PCR (qPCR), dPCR provides absolute quantification, often with higher precision at low copy numbers and reduced susceptibility to inhibitors. However, it generally offers a narrower dynamic range, higher equipment and reagent costs, and the need for specialized instrumentation. It is not typically used for broad, high-throughput expression profiling but is advantageous when precise measurement of low-abundance targets, copy number variation, or rare variants is required.

Common applications include analysis of copy number variation, detection of rare mutations and minority variants, quantification of viral or pathogen load, assessment of minimal residual disease in clinical samples, and quality control in research and diagnostic settings. Ongoing developments aim to increase partition numbers, improve multiplexing, and expand available platforms to broaden accessibility and throughput.