Digital PCR (dPCR) is a specialised technique that enables detection and precise, absolute quantification of target nucleic acids in a sample.
The technique shares similarities with qPCR but differs in two key aspects – the preparation of the PCR reactions and the method to determine whether DNA amplification has been successful. In dPCR, the sample is partitioned into thousands of independent PCR sub-reactions. Typically, the partitions are created using advanced microfluidic valving, microwells or the droplet digital PCR technique whereby emulsified microdroplets are suspended in oil. The BLINK approach utilises the BLINK Beads to provide the partitioning mechanism. This process randomly distributes the target molecules amongst the partitions so that each partition contains either a few or no target molecules. An optimal distribution results in no more than 50% of partitions containing the target.
The BLINK approach utilises the BLINK Beads to provide the partitioning mechanism.
Following distribution, each independent partition is then amplified and the end-point fluorescent signals are detected and interpreted to determine whether a partition contains the target sequence (positive) or not (negative). Absolute quantification of the target sequence is then calculated, based on the fraction of positive partitions. The accuracy of the quantification is defined using Poisson’s statistics, which models the random distribution of the target sequence into the partitions.
This technique doesn’t rely on calibration curves that are essential in qPCR. Here, quantification is based on binomial statistics (ie. a positive or negative outcome), thus converting an analogue signal into a series of digital signals. By removing the need for calibration curves, it removes variations in reaction efficiencies.