What’s the Digital PCR？
what is Digital PCR ?First described in the 1990s, digital PCR is a novel approach to PCR which allows precise detection and quantification of the amount of nucleic acids formed during PCR.
It is different from conventional PCR as it counts the target molecules directly in a digital format without relying on endogenous controls or standards. Thus, digital PCR finds use in many applications having limited sample availability and requiring high sensitivity.
Principles of Digital PCR
Digital PCR reaction is performed by diluting the sample and assay mixture into several thousands of small compartments. The basic concept behind this dilution is that the target molecule concentration in each reaction can be safely assumed as either zero or one.
At the end point, thermal cycling is carried out. Compartments that contain the target molecule will show fluorescence while the ones that do not contain the target molecule show only background fluorescence. Reactions with the target molecule are counted as 1 or PCR-positive reactions and the reactions without the target molecule are counted as 0 or PCR-negative reactions.
After counting the complete set of reactions, the number of target molecules in the entire reaction volume will be equal to the number of positive reactions counted. Therefore the absolute target concentration can be calculated by dividing the total number of target molecules by the total volume.
For the best possible accuracy, dPCR methods should have mechanisms to control errors in measured volumes and make sure there is not more than one target molecule in each compartment. Poisson statistics can be used to determine the chances of more than a single target molecule being present in one compartment. The higher the dilution and the number of replicates, the higher the sensitivity and accuracy of the dPCR analysis.
Thus, digital PCR is a third generation of PCR that employs a combination of sample dilution, end-point PCR, and Poisson statistics to achieve an absolute quantitation of the nucleic acid. This advanced technique builds upon traditional PCR and is ideal for applications requiring the detection of tiny quantities of nucleic acid sample such as copy number variation analysis, rare gene expression analysis, single cell analysis, and rare sequence detection.
Advantages of Digital PCR
The key advantages of digital PCR are listed below:
No dependency on standards or references
Linear detection of minute-fold changes
Capability to analyze rare events and complex mixtures
Highly inhibitor-tolerant
Allows increased precision by increasing dilution and target compartments.
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