Digital PCR Principle, Techniques and Types of PCR Applications
Digital PCR Principles
Digital PCR, the next generation of DNA amplification, is based on partitioning the fully assembled PCR mix into thousands of individual reaction compartments prior to amplification. Nucleic acid molecules are thus distributed across partitions, and amplified separately, alleviating any potential competition, and enabling absolute quantification of the DNA or RNA molecules.
This highly sensitive technology has already proven its reliability in applications such as Absolute Quantification (no need for standard curves!), Rare Event Detection (for example quantification of tumor-derived sequences in liquid biopsies), Gene Expression and miRNA quantification, exquisite discrimination in Copy Number Variation (CNV), and Next Generation Sequencing Library Quantification, amongst many others…
Digital PCR is fast becoming a mainstream technique in all labs, from academia to biotech and pharma to industry.
History of PCR
Since the invention of PCR by Kary Mullis in 1983, it has been a real revolution in molecular biology. A decade later, real-time PCR also termed quantitative PCR, offered the possibility of monitoring the PCR process. Until now, real-time PCR is considered as the “gold standard” for gene detection and quantification. But real-time PCR has its drawbacks such as the need for an external reference for quantification and a lack of sensitivity at low concentrations.
Introduction of Digital PCR
To further assess these drawbacks, Digital PCR was developed. The aim was to enhance the sensitivity of real-time PCR, especially in cases where detecting point mutations of low abundance in a background of wild-type DNA is required.
This technology is based on PCR amplification of DNA templates that have been randomly isolated into individual reactions. Each reaction contains zero, one or more molecules.
PCR amplification is then conducted using fluorescence dyes or probes, similar to real-time PCR.
The number of positive and negative reactions is counted by assessing the fluorescence intensity of each reaction at the end of the PCR reaction. For each reaction, a binary result is obtained explaining the term “digital”.
Finally, the proportion of positive and negative reactions is fitted into a Poisson distribution in order to obtain the absolute concentration of target molecules, eliminating the need for external references. Learn more about Poisson Distribution in Digital PCR in this detailed item: Poisson Law Computation.
A free calculator that automatically estimates target gene concentrations is also available here: Poisson Law: going further
The key feature of digital PCR is the compartmentalization of the sample. This isolates the DNA fragments from each other and then creates an artificial enrichment of low abundance sequences, leading to a higher detection level.
Finally, the dynamic range of the method is dependent on the total number of compartments generated.
Technologies to achieve a digital PCR
Until recently, digital PCR was performed using PCR plates. This process was labor-intensive and reagent consuming- limiting its use for routine analysis.
The latest advances in microfluidics enable massive automated partitioning of DNA sample in hundreds to millions of nano- or pico-liter scale compartments. Digital PCR array chips, splitting the sample in hundreds of microfluidic wells, combined with real-time PCR machines was the first digital platform developed.
An even higher throughput partitioning was obtained with a water-in-oil emulsion. Using microfluidic circuits and surfactant chemistry, thousands to millions of monodispersed partitions containing template DNA and PCR reagents can be generated, thermocycled, and then analyzed using automated partition flow-cytometers or scanners.
Applications of Digital PCR
Digital PCR is constantly proving its reliability in fields of application where precision and sensitivity are vital.
Thanks to its exquisite sensitivity, mainly required for the detection of point mutations, digital PCR met its first application in the field of oncology.
Wider applications in clinics and human biology were then developed including prenatal diagnosis, organ transplantation, and diabetes monitoring.
Digital PCR has proven its adaptability in numerous molecular assays and is now recognized as a reliable method not only for rare alleles and rare mutations detection but also for genotyping, absolute quantification, and copy number variation.
The areas of research where dPCR can be utilized are rapidly increasing. dPCR has been successfully implemented in the fields of virology, food and environmental microbiology applications, and for the monitoring of genetically modified organisms.