Digital PCR - Overview
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
Differences Between Traditional PCR and Digital PCR
The key differences between traditional PCR and digital PCR are tabulated below:
Traditional PCR estimates the amount of amplified PCR product at the end of the several PCR cycles.
Digital PCR measures the number of target molecules directly by counting positive fluorescence in compartments.
Traditional PCR is semi-quantitative at best. The concentration of nucleic acids can be determined to an extent by comparing the amplified band intensity on an electrophoresis gel to a known standard.
Digital PCR allows absolute quantitation of the target molecule using a unique blend of sample dilution and Poisson statistical algorithm.
The use of traditional PCR is limited to amplification of nucleic acids for sequencing, cloning, and genotyping
Digital PCR is highly sensitive and accurate and can be used effectively in absolute measurement of nucleic acids, rare gene detection, and absolute quantification of gene expression
Traditional PCR has low sensitivity and poor accuracy
In digital PCR, desired level of accuracy can be achieved by increasing the number of replicates
Traditional PCR is a non-automated process
Digital PCR can be easily automated for higher precision
Only size-based discrimination is possible in traditional PCR
Digital PCR can be used for accurate analysis of complex mixtures
No numerical expression of results and post-PCR processing is required in traditional PCR
Digital PCR provides user friendly results in digital format