The principle of dPCR explained – Absolute, not relative
dPCR video series - Episode 1
Digital tools are part of our daily life. But what makes PCR digital? And how is partitioning achieved in nanoplates? This video explains the principle of digital PCR in a simple way!
Digital tools are part of our daily life. But what makes PCR digital? And how is partitioning achieved in nanoplates? This video explains the principle of digital PCR in a simple way!
In dPCR, the nucleic acid sample is partitioned into numerous individual reactions, randomly distributing target molecules. Each partition has zero, one or many molecules. After end-point amplification, the fluorescence is measured. A reaction with no target molecule is counted as a 0/negative, and a reaction with one target molecule counts as a 1/positive. The initial copy number and concentration of the target molecule are calculated by applying Poisson statistics.
Standard qPCR is applied to bulk reactions, so a mutant target must compete with a wild-type background and be present in abundance to be detectable. To overcome this problem, digital PCR relies on the partitioning principle achieved in microfluidic nanoplates. Because of the random distribution and partitioning, the competition between the mutant and wild-type is significantly reduced, resulting in a fluorescence signal from even one target molecule/rare event in a given partition. Digital PCR also enables the simultaneous detection of multiple targets using a multiplex assay. It effectively detects rare events, copy number variations, small fold-change differences, or absolute amounts of nucleic acids without relying on standard curves, unlike standard qPCR.
Standard qPCR is applied to bulk reactions, so a mutant target must compete with a wild-type background and be present in abundance to be detectable. To overcome this problem, digital PCR relies on the partitioning principle achieved in microfluidic nanoplates. Because of the random distribution and partitioning, the competition between the mutant and wild-type is significantly reduced, resulting in a fluorescence signal from even one target molecule/rare event in a given partition. Digital PCR also enables the simultaneous detection of multiple targets using a multiplex assay. It effectively detects rare events, copy number variations, small fold-change differences, or absolute amounts of nucleic acids without relying on standard curves, unlike standard qPCR.
For more in-depth information about QIAGEN's digital PCR, visit: https://www.qiagen.com/applications/digital-pcr