Digital PCR Principle & Advantages
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- Опубліковано 9 лют 2025
- For more information, please visit our digital PCR learning center : www.gene-pi.com
One of the greatest challenges in molecular biology is the detection of a rare allele or mutant sequence lost in a dense background of wild-type sequences. This situation is often encountered in oncology for example when trying to detect tumor-derived mutant sequences, present in very small amounts in liquid biopsies.
In this animation, the mutant sequence is colored in red and the wild-type is colored in green. Finding the mutant sequence is a typical needle in a haystack problem!
When using standard real-time quantitative PCR, the mixture of DNA molecules is amplified in bulk in a reaction mixture containing PCR reagents, including a Taq polymerase, PCR primers and two types of probes, each specific for the wild-type or the mutant sequence respectively. The wild-type probe is labeled with a green fluorophore, the mutant probe is labeled with a red fluorophore.
During the PCR amplification, the probes specific for the mutant and wild-type sequences anneal to their respective targets and release a fluorescence signal.
However, in a bulk reaction, the signal of the mutant target is in competition with the signal of the wild-type one, and when the mutant sequence is present at a very low fraction, it may not even be detected.
To overcome this problem, Digital PCR relies on a basic principle: partitioning the sample prior to PCR amplification in order to isolate individual DNA molecules in different compartments.
Today, several techniques are employed to achieve the partitioning. This is either done in solid microchambers or done in an emulsion of microdroplets.
When the sample is partitioned, all DNA molecules are randomly distributed into a large number of partitions, such that only one or a limited number of DNA molecules end up in each partition. Even if mutant and wild-type sequences end up in the same droplet, the competition between the two is greatly reduced.
During the PCR amplification, a fluorescence signature is generated within each partition according to its DNA content. One targeted DNA molecule is sufficient to generate a fluorescence signal in a given partition.
Finally, the concentration of wild-type and mutant sequences in the sample is precisely estimated by counting the number of each type of partitions, classified according to the measured fluorescence levels: the non-fluorescent negatives, the greens,… the reds… and the double positives.
Digital PCR also enables the simultaneous detection of multiple targets using a multiplex assay.
Rigorously, a formula based on the Poisson law converts the counted negative and positive partitions into the target concentration of both the wild-type and mutant sequences.
Digital PCR is a powerful tool for the detection of rare events but can also benefit other assays such as copy number variation, by being able to precisely measure less than 2-fold differences in copy number, or absolute quantification of nucleic acids, since this technique does not have to rely on comparison with precalibrated standards. Digital PCR is also a great tool for genotyping single cells.
All those assays are currently applied to a wide range of applications such as oncology, infectious diseases, environmental testing, prenatal diagnosis, organ transplant, etc.
How do you want to use digital PCR?
Very good and clear explanation and animation makes this very easy to understand
great video! best explanation of digital PCR I've seen so far
Thank you! If you are interested in learning more about Digital PCR please visit www.gene-pi.com/
Very nice technology and very good explanation, bravo!
This explanation was great! Thank you so much!
What a great explanation, perfect for understanding this specially in Covid times.
very clear explanation！
Excellent video!
Why do you use fluorescence markers for the wild type allel in conventional qPCR if you are only interested in the mutation allel?
In qPCR, as for the digital PCR, the wild type strain is often considered a reference used to compared data relative to mutations.
The dPCR is a method which allows the absolute quantification of a template (DNA or RNA),
but it is also used to produce comparative (or relative) results, one example could be the necessity to compare the fraction of mutated sequences out of the wild type ones in a certain sample. At the following link www.gene-pi.com/tutorial/introduction-rare-mutation-detection-2-3/ you can find an example of an assay of this kind.
Very helpful and informative Thankyou
Where can i find the references?
Thanks for the great explanation!
Great tech, awesome video!
Why don't we construct a primer binding with the SNP therefore only the Mutant DNA would get amplified and we can visualize it
Of course ! This could be also one strategy.
Depending on what are your expectaction you can design the assay you prefer.
The naica® system offers the possibility to have multiple channels which could allow at the same time, and in the same experiment, the detection of different targets.
So you separate the sample before testing .?
Yes, the PCR mix is loaded in our chips chips and then is partitioned in thousands of small droplets in which the template sequences (DNA or RNA) will be randomly distributed.
For more information about our workflow, please check on our website here: www.stillatechnologies.com/naica-system-workflow/
Very nice video, i would be grateful if you could give me a reference of the Poisson law to convert the data into copies/µL/ Thank you very much !
Nice video! Does digital PCR also need to consider the effect of non-specific amplification caused by primer?
I appreciate
amazing! thank you!
Isn't this qPCR?
Thank you : )
Nice
Thompson Jose Martin Anthony Anderson Deborah