platform. The BioMark™ system was based on the 12.765 Digital Array, a chip of 12 panels, each panel partitioned into 765 6-nL chambers. However, digital PCR technology during this time was limited by the number of individual partitions per sample, by the volume of the reactions, and by increased hands-on time. Moreover, digital PCR was very costly to run, with a reaction costing several hundred dollars compared to a little over a dollar for traditional endpoint or real-time PCR.
The limitations of cost were addressed by the development of microdroplet-based lab-on-chip systems. These microfluidic systems partitioned samples into microdroplets that were generated by flow-focusing, rather than partitioning these samples into microchambers. The partitioning was then followed by thermocycling of the microdroplets, amplification, and end-point detection via real-time fluorescence curves. This technology was first commercialized by QuantaLife, Inc. as the QX100 ddPCR™ System in 2011. The microfluidic consumables used on the ddPCR™ platform could accommodate up to eight samples per chip, generating 14,000-16,000 droplets per sample.
These advances in digital PCR technology led to further expansion of its applicability in molecular biology and clinical genetics. However, the long hands-on time, low sensitivity, number of consumables required to run an experiment, and the limitation of multiplexing up to only two detection channels persisted.
The 2016 launch of the Naica™ Systema unique digital PCR platform with high sensitivity and precision, significantly reduced hands-on time, and a 3-color target multiplexing capability – marks a milestone in digital PCR technology innovation. By partitioning the sample into a large 2D array of droplets by confinement gradient, Crystal digital PCR™ combines the advantages of a) array-based digital PCR such as an integrated workflow and multiplexing; with b) those of dropletbased PCR such as reduced cost. The use of a confinement gradient to generate droplets ensures homogeneity in the droplet size, eliminates the need for oil flow, and the droplet size does not depend on the physical properties of the droplets (viscosity, surface tension, etc.).
The Crystal digital PCR™ technology can be used for nucleic acid quantification in a wide range of assays including, but not limited to, oncology (copy number variation, mutation detection, rare event detection, therapeutic monitoring5), infectious diseases (pathogen detection), food and GMO testing, Environmental testing, Gene editing and epigenetics, Neurobiology, NIPT. Absolute DNA and RNA quantification, Whole genome amplification, Droplet recovery, NGS library calibration/NGS result validation.
To learn more about digital PCR, designing assays for nucleic acid detection and quantification, statistical analysis, and the principles behind digital PCR experimental design, visit www.gene-pi.com