Illustration of the different fluorescence spectra in High Definition PCR.
High Definition PCR can expand the multiplexing of digital PCR tenfold.
iStock, Nobi_Prizue

Enhanced PCR Capabilities with Digital PCR

High Definition PCR can expand the multiplexing of digital PCR tenfold

May 17, 2022
Kristin Fathe, PhD
Photo portrait of Kristin Fathe, PhD
Kristin Fathe, PhD

Kristin Fathe, PhD, is a technical product manager at ChromaCode. She has more than six years of experience in the molecular diagnostics industry in various scientific and commercial roles. Kristin received her PhD in biochemistry from the University of Texas Austin, where she focused on the etiology of neural tube defects and completed post-doctoral training in pharmaceutics, specifically the delivery of biologics.

COVID-19 has forever changed lab expectations and now technology must play catch up. We expect rapid result turn arounds. We expect 24/7 digital access to our health care providers. The term PCR is being used in every day conversations. And we have seen the ramifications of inequitable access to diagnostics. The bottom line is that we now expect our collective experience to inform the future, and technology must answer the call.

In a new paper published in Analytical Chemistry, Jacky et al. describe the utility of a novel technology known as High Definition PCR, or HDPCR™, demonstrating that it can expand the multiplexing of digital PCR (dPCR) 10 fold. As a proof of concept, this was applied to human genomics using an aneuploidy model system.

The concept of multiplexing in a single-color channel using standard hydrolysis probe chemistry (TaqMan®) on a real-time PCR instrument helps when you need to detect targets as present or not present, as expected from a canonical real-time PCR assay. Things get a little different when looking for genomic features that require precise quantitation, like copy number variants or aneuploidies, for which you would need digital PCR (dPCR). Applying HDPCR to dPCR instrumentation results in thousands of reads from a single sample, thereby enabling absolute quantification of multiple targets. In the study, the goal was to determine relative amounts of chromosomal material from cell-free DNA, something that is usually achieved with an NGS approach.

HDPCR leverages multiple sets of primers and probes to target different sequences on a specific chromosome, all in a single-color channel, on a dPCR instrument. All these different sequences are probe limited to yield the same level of end point fluorescence, regardless of the target detected. As a reaction is separated into thousands of partitions, random pieces of cell-free DNA will make it into each droplet. The presence of the different sequences in a single droplet can be represented by a Poisson distribution—in the same way you could model the random entry of individuals into a store over time if you knew the overall average entrance. 

Because each droplet may contain anywhere from zero to N different targets, based on random partitioning of cell-free DNA, you may get anywhere from zero to N fluorescence units in each droplet. In fact, you will get a series of bell-shaped curves at different fluorescence intensities that represent the populations of positivity. Relative concentrations are then calculated to reveal small differences in chromosomal abundance from which you can tell if there are things like two or more copies of a chromosome present for a fetus in maternal blood.

Of course, summarizing this technology into a few sentences trivializes the additional inputs that are necessary for it to function. Our researchers and data scientists have created algorithms to standardize baselines, cross-talk, and instrument-to-instrument variations, as well as the ability for an interface to deconvolute all of this data. Learning more about this advanced technology can help prepare your lab for the future of research and diagnostics.