Digital PCR Principle & Advantages

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?

[RollerBaller]

Very good and clear explanation and animation makes this very easy to understand

[greta]

This explanation was great! Thank you so much!

[fryad]

Very nice technology and very good explanation, bravo!

[mspacey]

great video! best explanation of digital PCR I've seen so far

[chrspa]

Why do you use fluorescence markers for the wild type allel in conventional qPCR if you are only interested in the mutation allel?

[SevenPhotonsFilms]

What a great explanation, perfect for understanding this specially in Covid times.

[soura]

Why don't we construct a primer binding with the SNP therefore only the Mutant DNA would get amplified and we can visualize it

[wayne]

So you separate the sample before testing .?

[loicsavidan]

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 !

[geneluffy]

Nice video! Does digital PCR also need to consider the effect of non-specific amplification caused by primer?

[JosephLewis-ih]

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