Digital polymerase chain reaction (PCR) quantifies DNA by isolating and amplifying individual molecules and then counting templates individually as linear digital signals to provide a quantitative readout. Since its first description by Bert Vogelstein and Kenneth Kinzler in 1999, developers have taken advantage of rapid developments in the field of microfluidics to enable better separation of template molecules into nanoliter sample volumes. This has permitted smaller reagent consumption and increased the number of possible parallel digital PCR reactions that can be performed. Now, researchers at the State of Utah Center for Excellence for Biomedical Microfluidics have developed a new separation and amplification approach for digital PCR using a novel spinning disk platform, which could further advance efficiency and eliminate the need for labeled probes.
“The goal of the spinning disk platform is to reduce disposable and instrument costs from current digital PCR methods, and improve turnaround time by using rapid thermocycling techniques,” said Scott Sundberg, lead author of a new article describing the spinning disk digital PCR approach in the journal Analytical Chemistry.
The spinning disk is made of three thin sheets of plastic film, which are laminated together with an architecture that divides a sample into 1000 nanoliter-sized compartments during centrifugation. This eliminates the need for microfluidic valves and pumps to perform sample separation, and thereby reduces microfluidic loading time as well as disposable chip costs. PCR time is also reduced by performing reactions on the disk in an air chamber, which enables rapid temperature cycling. Following PCR, each compartment is evaluated for a DNA signal using the dsDNA fluorescent saturating dye LCGreen, which circumvents the need to use labeled probes.
The new platform was tested by amplifying plasmid DNA provided by the biological manufacturing company Lonza. The researchers reported that their platform completed the microfluidic digital PCR in less than 50 minutes, with an accuracy of 94%. Sundberg noted that real-time PCR reactions need to have an efficiency of 90% or greater to be of real use.
Although DNA sequencing technology is causing a shift in focus from PCR amplification to single-molecule sequencing, Sundberg suspects digital PCR approaches will continue to play a role in DNA analysis. “I believe PCR will still have a place in DNA sequencing for many years, although its role will most likely start to diminish,” he said. “Single-molecule sequencing is definitely a game-changing technology that may finally achieve the $1000 genome mark.”
“One nice distinction of digital PCR from areas such as sequencing is that it is really good at finding rare events [including] cancer detection, cancer therapy response, viral load, and prenatal testing,” said Sundberg. “This niche technology will become more and more useful as our understanding evolves.”
Sundberg and colleagues intend to develop the spinning disk platform for widespread use. “Currently, we are using a large benchtop prototype, which has validated the concept,” explains Sundberg. “Work is now underway to take [that] platform and develop a commercialized instrument.” One additional improvement Sundberg hopes to makes in the future is the incorporation of a fluorescent scanner to provide higher-resolution fluorescent imaging.
“The hope is that our low-cost platform will enable more researchers to become involved with digital PCR,” said Sundberg. “There are still many areas and methods to be investigated.”
The paper, “Spinning disk platform for microfluidic digital polymerase chain reaction,” was published Feb. 15, in Analytical Chemistry.