Digital PCR at GENERI BIOTECH

At our regular monthly Science Club, we recently explored the principles and applications of digital PCR (dPCR). Together, we reviewed not only the theoretical foundations and technological differences compared to established quantitative PCR (qPCR), but also presented initial findings from our own testing and development efforts.

Why dPCR?

While conventional quantitative PCR (qPCR) relies on measuring fluorescence in real time during the exponential phase of amplification, digital PCR approaches the problem from a fundamentally different perspective. Prior to amplification, the sample is partitioned into a large number of micro-reactions—either as droplets in droplet-based dPCR systems or via specialized microfluidic chip architectures. Fluorescence is then measured as an endpoint readout for each individual partition.

This technological shift provides several key advantages for laboratory applications:

• Absolute quantification: eliminates the need for calibration curves, enabling direct determination of the absolute number of target molecule copies.
• Ultra-high sensitivity: dPCR excels in detecting rare target sequences and mutations within a high background of wild-type variants.
• High robustness: partitioning into thousands of micro-reactions significantly mitigates the impact of PCR inhibitors.
• Broad applicability: the technology is invaluable for absolute viral load quantification, precise copy number variation analysis, and detection of complex molecular targets with multiple sequences.

From theory to practice: development at GENERI BIOTECH
At GENERI BIOTECH, we go beyond theoretical exploration. For our development experiments, we utilize the advanced QuantStudio Absolute Q system, which is based on innovative microfluidic array technology.

During the Science Club session, we provided insight into our optimization workflows. Encouragingly, we have experimentally confirmed full compatibility of our established assays (including, among others, the POLR2A assay) with dPCR technology and commercially available master mixes.

Our R&D team, however, is already looking ahead. We are optimizing parameters such as the ideal concentrations of reference dyes (e.g., ROX) to achieve the cleanest and most accurate signal readout possible.

Our future plans are clearly defined—and ambitious.

Magdalena Křišťanová

Leona Hofmeisterová