Digital PCR Explained: Precision, Sensitivity and Real-World Applications
Learn everything you need to know about digital PCR from an expert user.
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Digital PCR is a type of polymerase chain reaction (PCR) technique that was first described in the literature by Vogelstein and Kinzler in the late 90s. Prior to Vogelstein and Kinzler’s paper, several versions of digital PCR had already been developed, but were referred to as “limiting dilution PCR”.
Initially, digital PCR was not the easiest of methods for labs to perform; it was complex, time consuming and labor intensive. But, in recent decades, the commercialization of novel platforms simplifying digital PCR processes has resulted in it becoming a prominent technique in many labs, particularly those conducting clinical research. In 2023, the global digital PCR market was valued at $584.5 million and is expected to grow to $3,679 million by 2032.
The Genetics Core at the Edinburgh Clinical Research Facility is one example of a UK-based lab that routinely utilizes digitalPCR. The Core provides secure receipt, processing, archiving and analysis of biological samples, supporting the entire pipeline of clinical research from sample collection to genetic analysis.
Dr. Tamara (Tammy) Gilchrist is a research technician in the Genetics Core. In this interview with Technology Networks, Gilchrist offers a comprehensive overview of what digital PCR is, explains why researchers might opt to use it over other types of PCR and shares her tips for success in digital PCR experiments.
Can you explain what digital PCR is, and discuss its evolution as a technique?
Tamara Louise Gilchrist, PhD (TLG):
Digital PCR is a technique with many similarities to quantitative real-time PCR (qPCR) but that allows the absolute quantification of target molecules in a sample. This is done through the compartmentalization of one large PCR reaction into thousands of tiny, individual reactions. This might be using microwells, channels or oil droplets depending on the system.
We use a platform that uses microfluidics to produce up to 30,000 individual droplets called crystals on a self-contained consumable chip. The dispersal methods for digital PCR have developed and improved as the technique has gained interest to increase the number and quality of these partitions.
Similar to qPCR, the individual reactions in digital PCR will incorporate a fluorescent signal when the desired target is present by using TaqMan chemistry or intercalating dyes. However, instead of following the signal's progression during the reaction, in digital PCR each micro-reaction will be scanned at the endpoint and identified as simply negative or positive for the target. Positive droplets will have one or more copies of the target molecule. Established statistical calculations based on Poisson distribution are then applied by the system's software to determine the absolute amount of the target in the sample based on this number of positive and negative reactions. To ensure the accuracy of the calculation, it must be ensured that the volume of the sample in each partition is kept the same, as droplets with larger volumes would be more likely to contain more copies of the target, and thus the Poisson distribution is not comparable between droplets. The quantification uncertainty is also countered by increasing the number of partitions analyzed, such that any error in calculation is reduced.
Due to the differences in dispersal methods and also how the subsequent fluorescent signal is detected, different platforms that are available may be more suited for certain experimental applications, so this should be considered when you are planning experiments.
As the technique has developed, the available systems have also been improved to detect multiple targets from a single sample, such that now a vast range of multiplexing techniques have been described that extend the power of the technique massively.
MC:
Why might a researcher choose to conduct digital PCR over other types of PCR?
TLG:
A major reason to use digital PCR is to utilize the precision and sensitivity of the assay. As digital PCR requires no standard curve, you are therefore not extrapolating the concentration based on its correlation to known standards, you are directly calculating the concentration. This is regarded to be more precise and reproducible than qPCR.
Another advantage of using digital PCR is the way the sample is compartmentalized into many separate reactions. This approach increases the likelihood of detecting rare alleles or targets present in very low quantities, offering greater sensitivity and accuracy. Additionally, working in these very small amounts means any impurities in the reaction are generally better tolerated in digital PCR as potential PCR inhibitors will be diluted.
Most digital PCR systems also offer the opportunity to multiplex reactions, which allows the detection of multiple targets from a single, small volume of sample. This is particularly helpful if you have very precious samples of limited volume or concentration and increases the potential of your experiments dramatically.
MC:
Are there any limitations or challenges associated with digital PCR?
TLG:
One of the main challenges associated with digital PCR is designing effective primers and probes, particularly when you're planning to multiplex.
With multiplexing, you have the fantastic opportunity to detect multiple targets in a single experiment, but this advantage requires substantial assay development. This process can be very time-consuming and complex due to the precision needed in designing these components to prevent cross-reactivity and ensure accurate and reliable detection of each target.
Companies are addressing these challenges by introducing new products that include standardized universal fluorescent reporters and custom assays that can simplify the development process, making it more accessible and less daunting for researchers.
Another hurdle with digital PCR is that it demands specific – and often costly – capital equipment and consumables, which might not be available in all laboratories.
By offering digital PCR in our genomics core facility, we hope to make the equipment, and the expertise to run it, more accessible to researchers from different backgrounds and career stages.
MC:
TLG:
In our laboratory, we are most routinely using digital PCR to detect DNA that has been released into the bloodstream of a patient. This could be to detect DNA released from a tumor in a cancer patient, or it may be to detect an inflammation response in a patient with inflammatory bowel disease.
Using digital PCR to detect circulating tumor DNA (ctDNA) is particularly exciting as it allows the profiling of tumors using a non-invasive method. It can be used to identify therapy-resistant mutations and track disease progression even though the targets are in very low abundance compared to the background DNA.
We are also looking at using digital PCR in our next-generation sequencing (NGS) workflows, as this has the possibility of offering a more accurate quantification of NGS libraries, which means we can be more accurate when pooling different samples. We are still at the testing stage, as it is a tricky balance between the more accurate quantification offered by digital PCR and the additional cost compared to current standard techniques.
MC:
TLG:
I think it is most important to carefully design your digital PCR experiment based on the research questions that you want to answer. Consider the platform that you will be using, as they will each have specific benefits as well as certain limitations. In my experience, the running of the actual digital PCR experiment is the easy part!
As I mentioned before, taking time to optimize the primers and probes is very important, and this will pay dividends in the long run. This is especially true when multiplexing to ensure no cross-reactivity or quenching occurs.
Experimental contamination is always a worry as the assay is so sensitive. It becomes even more important to ensure that you use dedicated pipettes, tips with filters and conduct work in a clean area to help reduce potential issues. To ensure the best results, the equipment must also be calibrated and maintained correctly. Additionally, we ensure that in every run we include both positive and negative controls to help ensure the accuracy of the data.
Finally, keeping good documentation is key. The Digital MIQE Guidelines (Minimum Information for Publication of Quantitative Digital PCR Experiments) provide a standardized set of key information that should be collected and reported when publishing digital PCR experiments. Considering these at the experimental design phase will help to ensure that quality data is obtained from your digital PCR experiments.
