Digital PCR – a brief history

The groundbreaking discovery of the Polymerase Chain Reaction (PCR) by Kary Mullis in 1983 provided nearly limitless new opportunities for manipulation and detection of nucleic acids. In 1987, thermal cyclers became commercially available to perform PCR, which became an indispensable tool for molecular biology and clinical genetics.

In the early 1990s, several research groups began exploring the possibility of diluting the template DNA to an extent such that, on average, any single PCR reaction contained only a single template molecule, a method named “limit dilution PCR”. First described by Sykes et al. in 1992, this method had the major advantage of amplification of individual DNA molecules and it significantly reduced background noise1.

In a limit dilution PCR experiment, because of the multitude of reactions, the frequency of positive and negative reactions follows the Poisson distribution which can be used to calculate the abundance of target molecule based on the dilution factor. Limit dilution PCR was used to study, for example, disease burden in leukemia1, human genetic haplotyping2, and HIV provirus profiling3.

Vogelstein and Kinzler developed a strategy for selective amplification of rare mutations and distinguishing mutant from wild type DNA in colorectal cancer patient samples. The authors coined the term Digital PCR in 1999, denoting the similarity of classification of the individual reactions as “zeros/negatives” or “ones/positives” to bits in computer science4.

Digital PCR relies on partitioning a sample into numerous distinct components within which the PCR reactions then occur. The scientific community quickly recognized the unique advantages of digital PCR over traditional endpoint or real-time PCR: the assay does not rely on a standard curve; has improved accuracy; provides absolute quantification; and offers improved detection of low copy-number variants. Other key advantages of digital PCR became evident later as the method was used more broadly7: reproducibility over time and across different labs; robustness; and tolerance to PCR inhibitors.

The mid-1990s was a period of rapid development in microfluidic engineering and design. Biochemical assays could be performed on microfabricated chips, allowing partitioning, increased thermocycling efficiency in a smaller microenvironment, and an increase in the concentration of the reagents within the reaction mix. These lab-on-a-chip devices used miniaturized PCR components and did not require novel hardware, biochemistry, or DNA detection methods. Integrated DNA chips thermocycled reactions by transporting the mix through microcapillaries traversing heaters at the appropriate temperatures for DNA amplification.

Engineering and microfluidics drove further innovations in digital PCR, and in 2006 Fluidigm launched the Biomark™ system, the first commercial digital PCR 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 System- a 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 droplet-based 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

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Digital PCR History

References:

1. Sykes, P. J. et al. Quantitation of targets for PCR by use of limiting dilution. Biotechniques 13, 444–9 (1992). PMID: 1389177

2. Ruano, G., Kidd, K. K. & Stephens, J. C. Haplotype of multiple polymorphisms resolved by enzymatic amplification of single DNA molecules. Proc. Natl. Acad. Sci. U. S. A. 87, 6296–300 (1990). PMID: 1974719

3. Simmonds, P. et al. Human immunodeficiency virus-infected individuals contain provirus in small numbers of peripheral mononuclear cells and at low copy numbers. J. Virol. 64, 864–72 (1990). PMID:2296085 

4. Vogelstein, B. & Kinzler, K. W. Digital PCR. Proc. Natl. Acad. Sci. U. S. A. 96, 9236–41 (1999). PMID: 10430926

5. Madic, J. et al. Correction: EGFR C797S, EGFR T790M and EGFR sensitizing mutations in non-small cell lung cancer revealed by six-color crystal digital PCR. Oncotarget (2019). doi:10.18632/oncotarget PMID: 30647840