Digital PCR (dPCR) extends traditional PCR into the realm of real-time quantitative PCR. It is a method used to directly quantify and clonally amplify nucleic acids. It is an accurate and sensitive technique that counts individual nucleic acid molecules for applications such as detection of rare mutations and quantifying viral load, gDNA, cDNA, plasmids or next-generation sequencing libraries.

While conventional PCR methods rely on exponential detection curves, dPCR quantitation utilizes linear signals characterized as on/off or “digital” signals. The process begins by first diluting the sample in such a way that single DNA molecules are essentially isolated. This is done by producing a large number of aliquots and analyzing the reactions by traditional end-point PCR. Some wells of a microplate will contain a DNA molecule and produce an “on” signal, while other wells with no DNA material will produce an “off” signal. As a result, the user can calculate the number of positive and negative reactions to determine the absolute number of target molecules in the original sample.

This method has been utilized for more than a decade and has endured somewhat limited market success until recently. Fluidigm, an expert in microfluidic instrumentation, was the first to commercialize a chip-based dPCR system. Its BioMark system is a high-throughput, real-time dPCR instrument that can run thousands of PCR reactions without the use of robotics. The company purports that the system can produce 39,960 digital PCR reactions in the same amount of time as a single 384-well plate, while using just 1/200th the amount of reagents.

Life Technologies acquired several companies within the last year that are developing technologies for dPCR. It acquired two companies with expertise in microfluidics-based technologies for dPCR: Cytonix in 2009 (see IBO 7/31/09) and Stokes Bio earlier this year (see IBO 4/30/10). It also acquired BioTrove’s OpenArray Real-Time qPCR system (see IBO 11/15/09) and recently introduced its first kit to facilitate dPCR on this platform. Using the kit, researchers can perform over 9,000 dPCR reactions simultaneously and generate more than 36,000 data points per day.

Last month, QuantaLife announced its Droplet Digital PCR system, which includes a droplet generator and droplet reader. The system employs microfluidic technology to partition each sample into more than 20,000 one-nanoliter droplets, each of which contains a separate PCR reaction. The droplets are streamed in single file past a fluorescence detector to count the negative and positive reactions.

Digital PCR is a small but burgeoning technique within the $3 billion PCR market. With advancements in automation and microfluidic technologies as well as new product introductions, digital PCR has come of age and is quickly moving into the mainstream.

Digital PCR at a Glance:

Leading Suppliers

• Fluidigm

• Life Technologies

• QuantaLife

Largest Markets

• Academia

• Biotechnology

• Clinical/Diagnostics

Instrument Cost

• $50,000–$150,000