Ask me anything on dPCR? 

你問我答第三集

21. If I’m looking for rare DNA or have no idea about the DNA concentration I’m looking for, should I normalize the DNA concentration before the dPCR reaction?

Typically, using similar quantities of DNA facilitates the comparison between samples. However, when dealing with precious DNA samples, such as those from liquid biopsies, it might be preferable to omit this step to conserve the samples. In such cases, running a reference assay (or a wildtype control when seeking mutations) helps normalize variations in DNA amounts across samples

22. What’s the benefit of using dPCR in whole-genome sequencing (WGS) applications?

While WGS involves sequencing the entire DNA without targeting specific primers, dPCR requires primers and probes designed for known targets. Digital PCR is particularly useful in WGS studies for further investigation of specific targets identified during research. By designing specific primers and probes, researchers can use dPCR to verify and quantify findings from WGS studies. Additionally, dPCR is beneficial for accurately quantifying the libraries prepared for sequencing, which can help reduce costs by minimizing quality issues with the library.

23. Does digital PCR work for epigenetic activities?

The high sensitivity of dPCR can be very advantageous for epigenetic studies, where less sensitive methods can easily miss low-level expression detection of epigenetic changes.

24. How can dPCR distinguish rare events like mutations in one single nucleotide?

Measuring single nucleotide differences is a major application for dPCR especially in the clinical space. There are different approaches to achieve this. The one we use for our mutation detection assays uses one pair of primers but two different probes, one binding to the wildtype sequence and the other to the mutation/SNP. These probes then compete with one another. Since the probes have a higher chance of binding to their perfect match than to the other target (which has one mismatching nucleotide), the signal from the perfectly matching probe will be a lot higher than the signal from the mismatched probe. Both probes have a different fluorescent dye attached so you can count partitions that are positive for one target, the other target or even both and can calculate the copies/μl as well as the percentage of mutation.

When trying to screen for specific mutations, it’s also possible to run multiple assays using the same color probe, for example, when detecting the EGFR exon19 deletion with our pan-cancer assays. In this case, you won’t know the exact deletion, but you’ll be able to detect the presence of multiple possible sites at once with just one assay.

25. How can I use dPCR for CNV detection?

For CNV detection, the workflow is very similar to qPCR. Basically, you run one assay for your gene of interest and another assay for an endogenous reference gene of known quantity. You can also include a reference sample with known copies of your target per genome. Then you calculate based on either just the reference gene or the reference gene and reference sample. QIAGEN offers validated dPCR assays for thousands of targets on the human, rat and mouse genome, as well as custom CNV assays.

26. What is the minimum and maximum size of the fragments to amplify so that the calculation for copy numbers is still reliable?

The length of the amplified fragment mainly impacts the efficiency of the PCR reaction. This is less crucial in dPCR compared to qPCR since we are doing an end-point detection and only need efficiency that’s good enough to separate positive and negative partitions properly. Therefore, there’s no hard rule regarding fragment size. That being said, it’s advisable to use fragments of 60-150 bases, as even in dPCR, better efficiency means the assay will be more robust.

27. Can you tell us more about miRNA expression in vesicles and plasma samples with dPCR?

Digital PCR can be used to quantify miRNA in vesicles and plasma samples. An important consideration is to extract high-quality purified miRNA, which can then be combined with the necessary primers/probes for target detection and quantification. As miRNA expression can be relatively low, especially in plasma, dPCR offers high-level sensitivity and precision to detect low levels of miRNA and monitor their progression.

28.Are there applications of dPCR in infectious diseases?

PCR and dPCR can be used in research on infections and other diseases. Some of the applications include detecting rare virus particles or mutations in cancer patients, testing stool samples for the composition of the microbiome or applications in the production of biological compounds such as RNA vaccines. You can find a lot of interesting webinars on this topic, especially during our Digital PCR Festival in 2023.

29. Can you elaborate on the performance of dPCR in wasterwater analytics in terms of sensitivity, inhibitors and cost efficiency with low sample count per run (n=5)?

The QIAcuity has been used in several publications concerning the testing of wastewater and finding backtraces of pathogens. The limit of detection depends on a variety of factors (sample prep, target, assay used, amongst others). Generally, the limit of detection is as good or better than qPCR, with some customers claiming they can detect single molecules, with others being more in the 5 to 10 range of limit of detection. When it comes to PCR inhibition, we see fewer issues in the QIAcuity, particularly when using our QIAcuity Advanced One-Step Probe Kit, than we see in our qPCR tests. That being said, a high concentration of sample matrix can still affect the quantification, which is why we recommend testing different dilutions of samples. 

For cost-efficiency, we have an 8-well plate, so five samples and three controls would economically fill that up. In many cases, you need fewer technical replicates in dPCR than qPCR, so even if the per-well cost is higher in dPCR, the cost is potentially not as high as the cost of replicates.

30. What are the applications and limitations of dPCR for eDNA and aDNA?

Applications for the detection of environmental and ancient DNA are similar to qPCR. We have many projects with eDNA, including water surveillance, measuring microbes in soil or tracing marine organisms from water samples. The advantages of dPCR here are the increased sensitivity and tolerance to inhibitors. We have found that the QIAGEN One-Step Advanced kits perform really well with difficult sample matrices

31. What would be next big thing or new application area with this technology?

I can see a lot of dPCR being used in the growing market of cell and gene therapy as this includes the production of biological particles to carry genes/cells and during many of the steps in this production process, digital PCR can be used to control the quality of the product. Other emerging areas are wastewater-based epidemiology testing (remember COVID-19) and food testing (allergens, especially as they need to be detectable at really low levels). I can also see a lot of qPCR-based applications, such as clinical testing, especially in oncology, eventually switching over to dPCR. If you want to stay in the loop, I suggest you keep an eye on our dPCR webinar series.