Digital PCR-based assay: an answer to chromosomal abnormality
Digital PCR, also known as dPCR, was first described by Sykes et al. in 1992 (Sykes et al., n.d.), and although it has been around for a while, it is not as widely practiced as qPCR worldwide. Qualified as a “robust technique”, those who have made the switch appreciate the precision and sensitivity it offers. Particularly relevant for certain fields, it has emerged as a reliable technique for quantifying target sequences and identifying rare variants. It was therefore a logical choice for Stem Genomics’ founders back in 2018.
How does it work?
Digital PCR works just like qPCR, but the test is performed in many tiny droplets by partitioning and distributing the DNA and RNA randomly amongst them (1).
A single reaction takes place in thousands of nanoliter-sized droplets (ranging from 8 to 30,000 droplets depending on the platform used) (2). This distribution allows for multiple simultaneous endpoint PCR amplifications through the measurement and counting of positive (i.e. containing the amplified target) and negative (i.e. without the amplified target) partitions. The term ‘digital’ refers to this binary result counting method. Positive reactions, which contain at least one copy of the target DNA fragment, are identified by their fluorescence signal (3). The comparison of the number of positive and negative reactions enables target quantification using the Poisson distribution method (4).
Fields where dPCR is particularly relevant
Digital PCR is ideal for complex samples with a low concentration of target molecules, making it very popular in biopharma applications, oncology and organ transplantation fields.
It is also the most accurate PCR method for assessing the genomic stability of stem cells. Digital PCR can achieve a sensitivity of up to 0.1% (in certain situations even up to 0.001%), compared to 1% for the traditional PCR method (Lambrescu et al., 2022). Stem Cell research scientists use it to detect genetic variants of various sizes, including infra-karyotypic ones. Thanks to the intrinsic properties of the dPCR technology, high precision in detection and quantification is achieved.
Due to its remarkable analytical sensitivity, digital PCR has become a promising tool for accurate measurement of biomarkers that monitor disease progression and quantify therapeutic efficiency and can be considered a tool for non-invasive prenatal diagnosis and new-born screening (Lambrescu et al., 2022).
Digital PCR is expected to grow in importance as precision medicine becomes more commonplace.
Why do Stem Genomics clients choose to add our digital PCR tests to their workflow?
The main reasons are performance, speed and convenience. This is a complete contrast with G-Banding which, although it is the recognized method for checking genomic stability, presents many challenges in those areas for most research teams.
A typical use case amongst our clients is a team that already has a quality process in place, but that wants to go up a level in terms of performance, without generating delays and prohibitive expenses. DiNAQOR is a good example to illustrate this. According to Kurt Jacobs, a research scientist, “Stem Genomics’ assay covers a wide range of mutations in a simple test. It helps us strengthen our quality control at various stages of our workflow. For instance, it picked up the 20q amplicon that was present in some of our hiPSC lines.”
Our digital PCR-based assays also make regular in-process testing possible every 5-10 passages. This is a great help in detecting genomic abnormalities earlier in the process compared to G-Banding karyotyping only. According to Angélique Fourrier, R&D project manager at GoLiver, “since we started using the iCS-digital™ PSC for routine control, we have gained a massive degree of confidence in the quality of our cells in long-term culture, as we can quickly control their genome integrity even on a weekly basis if we wanted”.
Another very frequent scenario we have is that of clients looking to rapidly screen clones after gene editing. It provides a first level screening, which is fast and cost effective, before selecting the clones that will go through further genomic stability testing.
There is also the use case of teams that do not have sufficient funds available for thorough testing but still want to implement some control process. Although this is not the case in her lab, which has an extensive array of tests in place, Sandra Segeletz, Head of Innovation at denovoMATRIX, says “I would recommend the Stem Genomics tests to anyone looking for a good genomic appraisal at minimum effort. This is an easy entry to quality control for anyone with a limited budget.”
Views from the lab: Juline Vincent, R&D Project Manager at Stem Genomics
What is your background?
I have a Master’s degree in biotechnology health project management and experience in the fields of oncology and cell therapy.
Can you tell us more about your experience with dPCR? When did you first try it and how did it complement the technologies you had available in your lab?
My first experience with dPCR dates back to 2018 in the oncology field. I first started on a BioRad QX200 and got trained up by their company to use the machine. Later on I expanded my skills to other platforms. I was trying to detect cell free DNA, and as it is not very quantifiable, a technology like digital PCR was really interesting. For qPCR, it was like trying to find a needle in a haystack, so going digital was an obvious strategy for us. Just like it is here for Stem Genomics, where CNV detection requires highly sensitive technology.
Now that you work for Stem Genomics, what are the technical advantages that dPCR brings your clients?
Beyond the high precision for low-frequency mosaic abnormalities, there are many advantages in using digital PCR as a lab technician.
The first one that springs to mind is that you don’t have to worry about multiplexing. This saves you a lot of time and money at the end of the day. And for our kit users, having just one well instead of several makes a big difference.
Although we do it in our lab for additional precision, unlike qPCR, you don’t technically have to run replicates, as digital provides absolute quantification of nucleic acids without having to rely on external references (e.g. standard calibration curves).
It also gives robustness to variations and a high level of reproducibility. It dramatically reduces inter- and intra-assay variability and is less prone to cross-contamination than qPCR thanks to the sample dispersion method.
Finally, for clients whose samples are scarce, only a small amount of material is required to perform the experiments.
What will be the next developments for Stem Genomics’ clients?
Our focus is on continuously improving the performance and speed of our services and kits. We strive to stay on top of the latest industry R&D and regularly test new strategies. We are also actively working on the expansion of our services to other dPCR tests beyond genomic stability. And there is a lot going on at the moment in the digital PCR industry, which makes my job even more exciting!
Discover Stem Genomics range of digital PCR assays:
Our digital PCR services Our digital PCR kits
For those who don’t have a dPCR machine For those who have access to a dPCR machine
Sykes, P. J., Brisco, M. J., Hughes, E., & Morley, A. A. (n.d.). Quantitation of targets for PCR by use of limiting dilution Minimal residual disease in leukemia View project Parthenolide and prostate cancer View project. https://www.researchgate.net/publication/21766890
Lambrescu, I., Popa, A., Manole, E., Ceafalan, L. C., & Gaina, G. (2022). Application of Droplet Digital PCR Technology in Muscular Dystrophies Research. In International Journal of Molecular Sciences (Vol. 23, Issue 9). MDPI. https://doi.org/10.3390/ijms23094802