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Digital PCR (dPCR)

What is Digital PCR?

Digital PCR (dPCR) is an advanced technique for the precise quantification of nucleic acids, allowing detection and measurement of DNA or RNA molecules with exceptional sensitivity and accuracy. Unlike traditional quantitative PCR (qPCR), which measures DNA amplification in real-time, dPCR partitions the sample into thousands to millions of individual reactions.

Each partition contains either zero or one (or more) target molecules, resulting in a binary (digital) readout positive or negative. This allows absolute quantification without the need for standard curves and offers enhanced sensitivity, especially for rare targets or low abundance nucleic acids.

How Does Digital PCR Work?

  1. Partitioning
    The sample is divided into thousands to millions of separate micro-reactions, typically droplets suspended in oil (droplet dPCR) or tiny wells on a microfluidic chip.
  2. Amplification
    PCR amplification occurs independently in each partition. If the target molecule is present, amplification produces a detectable fluorescent signal.
  3. Detection
    After amplification, each partition is analyzed to determine if it is positive (fluorescent) or negative (non-fluorescent).
  4. Quantification
    Using Poisson statistics, the absolute number of target molecules in the original sample is calculated from the proportion of positive partitions, eliminating the need for calibration standards.

Advantages of Digital PCR

  • Absolute quantification without calibration curves.
  • High sensitivity and precision, ideal for detecting rare mutations or low copy number targets.
  • Robust against PCR inhibitors found in complex samples due to partitioning.
  • Multiplexing capability for simultaneous detection of multiple targets.
  • Improved reproducibility compared to qPCR, especially in challenging samples.

Common Applications

  • Rare mutation detection in oncology, such as circulating tumor DNA (liquid biopsies).
  • Viral load quantification for HPV, CMV with improved accuracy.
  • Copy number variation (CNV) analysis in genetics.
  • DNA methylation studies in epigenetics. 
  • Validation of qPCR results in research and diagnostics.

Technologies and Platforms

  • Droplet Digital PCR (ddPCR): Uses thousands of oil-water emulsion droplets, each acting as an independent PCR reactor.
  • Chip-based Digital PCR: Sample is partitioned into thousands of micro-wells on a chip.
  • Microfluidic devices: Utilize channels and chambers to partition samples for high-throughput analysis. 

Digital PCR is a powerful tool for absolute, sensitive, and precise quantification of nucleic acids, revolutionizing research, clinical diagnostics, and biotechnology. Its ability to detect low-abundance targets with high confidence is crucial for applications ranging from cancer genomics to infectious disease monitoring and environmental biology. By complementing and surpassing traditional PCR methods, digital PCR enables new discoveries and innovations across life sciences.