In 1971, a paper was published in the Journal of Molecular Biology that first described the process of using heat-resistant enzymes together with primers to replicate a short DNA sequence in vitro. The actual invention of the polymerase chain reaction (PCR) came over a decade later, in 1983. PCR was a revolutionary biochemical technique that allowed scientists to replicate and amplify nucleic acid sequences in millions to billions of copies.

Sequencing, cloning, and analysis were no longer limited by tiny amounts of samples available for testing.

Several different PCR forms were developed through further refinement of this process, including the digital polymerase chain reaction (dPCR). Unlike other PCR variants, dPCR allows for absolute quantitation of the target DNA sample, opening new doors for scientific research and discovery.

Absolute Quantification

The hallmark of dPCR (and droplet digital PCR, as one of its subtypes) is absolute quantification. Conventional PCR methods are only semi-quantitative – their final product has to be detected by agarose gel electrophoresis. Real-time PCR (qPCR) is relatively quantitative – it requires the use of standard curves and extrapolation based on patterns to calculate the target concentration of the nucleic acid in the original sample.

Digital PCR doesn’t need any referencing or extrapolation. After the amplification reactions are complete, it precisely and discretely measures the number of target molecules present at the beginning. This is achieved through massive partitioning or dividing the sample into thousands of individual compartments (droplets) that do not compete with one another. Each compartment represents a PCR reaction on its own. dPCR, therefore, offers thousands of data points instead of only one single result, leading to higher quantification accuracy.

The Principles of ddPCR Technology

Droplet digital PCR has emerged in the scientific community only in the last decade or so. It represents one of the most sensitive, precise, and accurate methods for amplifying and quantifying sequences of different nucleic acids (DNA, RNA, and cDNA).

ddPCR is based on a water-oil emulsion droplet system. The massive partitioning required for this type of PCR is performed in a single step thanks to a combination of surfactants and microfluidic technology. The ideal number of independent partitions or nanoliter-sized droplets in this process is twenty thousand. A single partition should contain zero or one template molecule (but no more than a few).

After the amplification, each droplet is analyzed with a fluorescent probe. If it contains at least one target copy, it is a positive droplet (fluorescent droplet). If it doesn’t contain any target copies, it is a negative droplet (exhibiting little to no fluorescence). This ratio of positive droplets to negative droplets is then analyzed with Poisson statistics to determine the concentration of the DNA template in the original sample.

ddPCR Workflow

Before a ddPCR experiment is performed, it is essential to understand the goal or the desired outcome of the experiment. Different experiment types require different amounts of target sequences, different preparation methods for the sample, and different kinds of data analysis. If it is well-designed and properly optimized, there is no reason for a PCR reaction not to yield robust results that can easily be reproduced.

Because ddPCR focuses on end-point analysis to generate the required quantitative data, it doesn’t depend on amplification efficiency as much as qPCR. Nevertheless, special care should be taken in assay design and optimization to allow for a clear differentiation between positive and negative droplets.