What is digital PCR?

[Mehreen A]

What is digital PCR and how is it different from quantitative PCR?

[Dr. Asim]

Digital PCR (dPCR) and quantitative PCR (qPCR) are both techniques for detecting and quantifying nucleic acid. The key difference between the two technologies is precision power. While both offer highly sensitive and reliable nucleic acid detection and quantification, dPCR provides precise, binary results by literally counting the presence or absence of DNA molecules. This clarity of results combined with a low error rate makes for an incredibly high level of precision. In contrast, qPCR is reliable but requires optimization to get a good result, and even then, you must contend with background noise. dPCR is well-suited to measure smaller quantitative differences and is more robust in the presence of inhibitors such as humic acid and heparin.

In summary, dPCR offers higher precision and robustness, while qPCR is valued for its speed, sensitivity, specificity, and ease of use.

 

[Hamdy Ahmad]

This is an invitation for a webinar on this subject:

Webinar: Digital PCR Automation With Flexibility to Scale | Thermo Fisher Scientific – EG

 

[A. Hammouda]

Instead of performing one reaction per well, dPCR involves partitioning the PCR solution into tens of thousands of nano-liter sized droplets, where a separate PCR reaction takes place in each one. The partitioning of the sample allows one to estimate the number of different molecules by assuming that the molecule population follows the Poisson distribution, thus accounting for the possibility of multiple target molecules inhabiting a single droplet.

dPCR measures the actual number of molecules (target DNA) as each molecule is in one droplet, thus making it a discrete “digital” measurement. It provides absolute quantification because dPCR measures the positive fraction of samples, which is the number of droplets that are fluorescing due to proper amplification. This positive fraction accurately indicates the initial amount of template nucleic acid. Similarly, qPCR utilizes fluorescence; however, it measures the intensity of fluorescence at specific times (generally after every amplification cycle) to determine the relative amount of target molecule (DNA), but cannot specify the exact amount without constructing a standard curve using different amounts of a defined standard. It gives the threshold per cycle (CT) and the difference in CT is used to calculate the amount of initial nucleic acid. As such, qPCR is an analog measurement, which may not be as precise due to the extrapolation required to attain a measurement.

dPCR measures the amount of DNA after amplification is complete and then determines the fraction of replicates. This is representative of an endpoint measurement as it requires the observation of the data after the experiment is completed. In contrast, qPCR records the relative fluorescence of the DNA at specific points during the amplification process, which requires stops in the experimental process. This “real-time” aspect of qPCR may theoretically affect results due to the stopping of the experiment. In practice, however, most qPCR thermal cyclers read each sample’s fluorescence very quickly at the end of the annealing/extension step before proceeding to the next melting step, meaning this hypothetical concern is not actually relevant or applicable for the vast majority of researchers. dPCR measures the amplification by measuring the products of end point PCR cycling and is therefore less susceptible to the artifacts arising from impaired amplification efficiencies due to the presence of PCR inhibitors or primer template mismatch.

qPCR is unable to distinguish differences in gene expression or copy number variations that are smaller than twofold. On the other hand, dPCR has a higher precision and has been shown to detect differences of less than 30% in gene expression, distinguish between copy number variations that differ by only 1 copy, and identify alleles that occur at frequencies less than 0.1%.

 

Source: Digital polymerase chain reaction – Wikipedia

 

 

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