Digging Out DNA with Digital PCR
The polymerase chain reaction (PCR) plays a leading role in today’s biology. PCR started with an endpoint approach that detected a particular nucleic-acid sequence. Then, real-time PCR provided relative quantification of the sequences. Most recently, digital PCR (dPCR) allowed scientists to absolutely quantify sequences of nucleic acids. This article explores dPCR techniques from Bio-Rad, Fluidigm, Life Technologies, and RainDance Technologies.
Some form of PCR can be found in almost any life science lab. As Paco Cifuentes, director, product applications at Life Technologies, says, “PCR is the most used technology in molecular biology for many applications.” For example, dPCR very precisely quantifies small changes in gene expression, copy number or the presence of any given molecule of DNA or RNA.
In addition, Howard High, Fluidigm spokesperson, says that “the technology’s extreme sensitivity in rare-event detection” can reveal “the presence of low-level foreign DNA in a high background of native DNA.” As examples, he mentions detecting fetal DNA in maternal blood or detecting genetically modified seeds where they should not be.
Moreover, Viresh Patel of Bio-Rad’s Digital Biology Center points out that rare-sequence detection can be used to look for a pathogen or virus in a patient. “This has been used for detecting very low levels of HIV in a person’s blood,” he says.
Rena McClory, director, digital PCR at RainDance Technologies, adds, “As our understanding of the low level variation that impacts disease state and progression grows, so too does the need for technologies with the capabilities of absolute quantification of these low-level DNA targets.” That’s just what dPCR can provide.
In essence, dPCR divides a sample into thousands of subsamples and performs PCR on each of them. The samples get divided into wells or droplets. Both approaches open doors to expanding applications of dPCR.
Cashing in on the Chips
For chip-based dPCR, Life Technologies developed the QuantStudio 3D Digital PCR System. “The consumable chip includes 20,000 partitions,” Cifuentes says, “and each is a chamber for an individual PCR reaction.”
With this platform, a researcher mixes a sample with its assay components and a master mix. That mixture is dispensed onto the chip and placed in a thermal cycler to run the PCR. Next, the chip goes in the QuantStudio 3D Digital PCR System, which reads the results in less than a minute. “You can have absolute quantification in copies per microliter, percentage of the rare event, copy-number, etcetera,” Cifuentes says.
For particularly rare sequences, a scientist can divide a sample over multiple chips, and the software can aggregate the data as if it were one chip. In addition, a Life Technologies adapter lets a thermal cycler simultaneously handle 24 chips.
Fluidigm takes another chip-based approach to dPCR. For example, the EP1 system provides endpoint reads. Conversely, the BioMark HD system collects the PCR data after every cycle. The Fluidigm systems can be used with two different integrated fluidic circuits, or chips, including the qdPCR 37K Integrated Fluidigm Circuit. High says, “This chip when used with the BioMark HD System combines both digital and quantitative real-time PCR.” He adds, “The qdPCR 37K IFC performs at a 99.9 percent success rate, providing ultra-high precision and throughput without the concern of false positives.”
Dividing with Droplets
The Bio-Rad approach to dPCR divides a sample into droplets. This company’s QX100 Droplet Digital PCR system starts with a 20-microliter sample that is prepared for PCR. Then, a consumable cartridge converts the 20 microliters to 20,000 nanoliter-sized droplets. The droplets go in a 96-well plate for PCR, and the QX100 reads the results drop by drop. “The system consists of a droplet generator and droplet reader,” Patel says. “The reader interrogates each droplet, determines the fluorescence amplitude, counts positive and negative droplets—ones that includes the target sequence and not, respectively—and calculates the concentration of the target nucleic acids.” Bio-Rad also produces super mixes for DNA and RNA detection and reagents that can be used with the QX100 to prepare DNA libraries for next generation sequencing.
RainDance Technologies also uses a drop-based approach. McClory says, “RainDance’s RainDrop System shifts the PCR paradigm from a single color per marker to a more scalable and precise multicolor and intensity-per-marker method.” This system generates 5–10 million picoliter-size droplets. McClory adds that this system can “detect one mutant amongst 250,000 wild-type molecules with a lower limit of detection of one in more than 1,000,000.” She adds that researchers can “conduct 10 tests or more on the same sample using the single molecule multi-color detection technique.”
The advances in dPCR spawn new applications, such as sample preparation for next generation sequencing. Other applications will emerge as scientists develop more uses of quantifying nucleic acids.