Digital PCR

For more information on digital PCR please contact Tammy Gilchrist or Lee Murphy

Digital PCR (dPCR) is an end-point PCR method that offers an alternative to conventional real-time qPCR for absolute quantification of nucleic acids and rare allele detection. The method is similar to qPCR in terms of the reaction components, where primers and labelled probes detect sequence specific targets. The subsequent amplification is also the same, however, digital PCR differs in the way the sample target is measured. In dPCR the nucleic acid sample mix is partitioned into many individual PCR reactions (droplets or wells). Then amplification of the individual reactions is conducted and each well or droplet will be either positive (1) or negative (0) for the target molecule.  The number of positive vs negative reactions is determined and permits the generation of an absolute count of the number of target molecules in the samples. This simple and reproducible method does not rely on a calibration curve for sample target quantification; therefore, no reference standards or endogenous controls are needed, allowing for greater robustness and reproducibility among different labs. Poisson correction is applied to account for individual reactions that may have more than one molecule of target sequence present. 

In the Naica System, digital PCR is integrated into a single consumable chip. The sample in each port (4 or 16 samples per chip) is partitioned into a 2D array of approximately 30,000 or 15,000 individual droplets of homogenous size by confinement gradient using the Naica Geode. We have 2 of these instruments, so can run up to 96 samples at once. Thermocycling is also performed on chip, for each droplet, in the geode immediately after crystal generation. Lastly, the chips are scanned using the Prism6 instrument and Stilla Technologies Crystal Reader Software using up to six different fluorescent channels. This allows multiplexing for numerous targets per sample, covering a diverse range of applications. The Crystal Miner software automatically measures the concentrations of targeted nucleic acids in each channel and allows clear visualisation of the data.