Digital Droplet PCR 

In life sciences, ddPCR is often chosen over qPCR (quantitative real‑time PCR) when absolute quantification, rare‑event sensitivity, and high precision are critical. The microfluidic ddPCR workflow typically unfolds in four steps:

1. Sample Partitioning 
Partitioning is achieved by using microfluidic droplet generators. The PCR mix — containing DNA or RNA, primers, probes, and enzymes — is separated into thousands of tiny microreactions. Each droplet may hold zero, one, or a few target molecules, creating a highly precise setup for amplification.

2. PCR Amplification 
Once partitioned, each droplet undergoes PCR independently. This may happen in tubes or wells in which the droplets are loaded from the droplet generator – or on the microfluidic chip itself.

3. Detection and Droplet Reading 
After amplification, droplets are scanned for fluorescence. A specialized droplet reader then examines every droplet to determine whether the target sequence is present, using fluorescence as the signal. Positive droplets glow to indicate the presence of the target sequence, while negative droplets remain dark. This binary readout makes digital droplet PCR highly accurate.

4. Data Analysis
Each droplet can contain one or several target molecules, therefore the absolute number of target molecules is calculated using the Poisson distribution. This calculation provides the absolute quantification of nucleic acids, eliminating the need for standard curves and ensuring unmatched precision in genetic analysis.