Droplet Digital PCR (ddPCR™) Frequently Asked Questions

Droplet Digital PCR technology is a digital PCR method utilizing a water-oil emulsion droplet system. Droplets are formed in a water-oil emulsion to form the partitions that separate the template DNA molecules. The droplets serve essentially the same function as individual test tubes or wells in a plate in which the PCR reaction takes place.

In traditional PCR, a single sample offers only a single measurement, but in Droplet Digital PCR, the sample is partitioned into 20,000 nanoliter-sized droplets. This partitioning enables the measurement of thousands of independent amplification events within a single sample.

Droplet Digital PCR (ddPCR) stands out as the superior choice among digital PCR technologies due to its track record spanning over 12 years. With more than 8,000 peer-reviewed articles supporting its usage, ddPCR technology has garnered a robust reputation in the scientific community. The key lies in its unique and patented partitioning method, which generates thousands of droplets per sample, ensuring reliable and consistent results. The unrivaled precision and accuracy of ddPCR technology have been thoroughly tested and validated by Australia’s National Measurement Institute (NMI) and other metrology institutions globally using DNA standards, solidifying its position as the gold standard for quantification and paving the way for groundbreaking research in various fields.

The Droplet Digital PCR (ddPCR) workflow follows a straightforward process. Initially, the Droplet Generator partitions samples into numerous nanoliter-sized droplets. These droplets undergo PCR on a thermal cycler, after which they are read by the Droplet Reader to count positive and negative reactions. By applying Poisson distribution to counted PCR-positive and PCR-negative droplets, the system delivers precise absolute quantification given as a concentration of copies per volume.

QX ONE™ ddPCR System Automated Walk-Away Workflow

QX200™ & QX600™ ddPCR Systems Workflow

Droplet polydispersion can affect accuracy in quantitation because of elevated Poisson distribution unpredictability. However, Droplet Digital PCR systems are not susceptible to that because a) the microfluidic chip architecture is engineered for droplet monodispersion and uniformity, and b) droplets that are too small or too large are gated out at the droplet reading stage. Bio-Rad’s heritage of chemistry engineering and optimization for stable and uniform droplets, as well as validation and high-quality manufacturing of products like supermixes, consumables, and reagents ensures uniform droplets and maximum sample utilization. This means the final data output comes from high-quality, uniform droplets.

Bio-Rad's ddPCR technology generates 20,000 droplets per 20 µl sample, nearly two million partitioned PCR reactions in a 96-well plate, the greater number of partitions yields higher accuracy. The accuracy and precision of the digital PCR results are tied to the sample partitioning volume according to the Poisson distribution. The following formula demonstrates how precision and accuracy are calculated:

Amidst the many digital PCR solutions in the marketplace, the focus on dead volume has become crucial, especially when dealing with limited sample volume or low copy number targets. Dead volume refers to the unutilized portion of the sample, lost during collection, preparation, and analysis stages of dPCR. Neglecting the early workflow steps of sample collection and preparation can lead to the loss of valuable sample volume and target template copies. Regardless of the dPCR platform being used, optimizing these steps is essential.

In the dPCR workflow, each platform in the market partitions, amplifies, and analyzes the sample. However, Droplet Digital PCR (ddPCR) technology. revolutionizes the process through emulsion-based partitioning, assay optimization, and unique gradient capabilities in the PCR amplification step. Droplet Digital PCR Systems create up to 20,000 sample partitions, ensuring avoidance of under sampling errors, regardless of consumable choices. Additionally, the gradient capabilities in ddPCR technology enable faster PCR optimization, conserving precious samples during optimization steps — a crucial consideration often overlooked by others.

Understanding digital PCR dead volume becomes especially crucial in scenarios with limited samples or applications involving rare targets. To optimize outcomes in such situations, it is imperative to consider the entire dPCR workflow and choose a platform, like Bio-Rad ddPCR technology, that maximizes the potential of your valuable samples with precision and reliability.

This can be true, depending on the degree of droplet coalescence. However, this is a phenotype that is seen infrequently and is easily mitigated by eliminating environmental debris in the manual PCR reaction setup, Bio-Rad has spent over a decade optimizing ddPCR technology to avoid coalescence or shearing of droplets.