What dPCR is?
Digital PCR (dPCR) is a quantitative PCR method that provides a sensitive and reproducible way of measuring the amount of DNA or RNA present in a sample. This method is similar to qPCR in the reaction assembly components and amplification reaction, but differs in the way the sample target is measured. In dPCR, the sample is first partitioned into many independent PCR sub-reactions such that each partition contains either a few or no target sequences. After PCR, the fraction of amplification-positive partitions is used to quantify the concentration of the target sequence with a statistically defined accuracy using Poisson’s statistics. Digital PCR is a simple and reproducible method that does not rely on a calibration curve for sample target quantification. No reference standards or endogenous controls are needed.
How dPCR works?
The initial dPCR reaction is assembled using the same reaction components as those used in qPCR. The subsequent step partitioning the sample reaction mix into a large number of individual wells is unique to dPCR. The method relies on the assumption that sample partitioning will follow a Poisson distribution resulting in 0 or 1 target per well. Upon completion of sample partitioning, PCR amplification reactions are run to endpoint. The presence or absence of fluorescence in the amplified reaction wells is then used to calculate the absolute number of targets present in the original sample. Wells with fluorescent signal are positives and scored as “1”; wells with background signal are negatives and scored as “0”. Poisson statistical analysis is then used to determine the absolute concentration of target present in the initial sample.
Step 1 – The initial reaction is assembled in a single tube using the same components as qPCR
Step 2 – Assembled reaction is split into a large number of individual wells resulting in either 1 or 0 targets/well
Step 3 – PCR amplification is performed to endpoint
Step 4 – Absolute quantification of target molecules is calculated using Poisson statistical analysis
Applications showing improved results using dPCR
Digital PCR has been shown to be advantageous for applications requiring high sensitivity, accurate quantification, reproducible quantification and improved multiplex capability.
- Rare allele detection
- Copy number variation
- Pathogen detection
- Quantification of Next-Generation sequencing libraries
- Gene expression heterogeneous samples
- Viral load detection