What is Digital PCR?
Digital PCR is an alternative method to qPCR and has similar characteristics. Digital PCR (dPCR) is a highly sensitive approach for nucleic acid quantification. It is a widely used method for nucleic acid detection. Instead of relying on the number of amplification cycles, as in qPCR, to determine the initial amount of the target molecule in the sample, this method estimates the absolute number of target molecules by statistical methods. Digital PCR is a highly sensitive approach for precise and reproducible nucleic acid detection and quantification. Measurements are performed by partitioning the sample so that ‘zero’ or ‘one’ target molecule is present in any individual reaction. Each section is analyzed after the endpoint PCR cycle for the presence or absence of a fluorescent signal and the absolute number of molecules present in the sample is calculated. The sample does not require a standard curve for target quantitation. The absence of a standard curve reduces the margin of error and increases precision.
How does it work?
The test components used in the dPCR reaction consist of master mix, primers, TaqMan probes and target DNA, as in qPCR.
It performs the analysis by splitting or dividing each sample into a large number of separate and parallel reactions. The master mix with the diluted sample is split and distributed into individual PCR reactions. The distribution of molecules within the sections is an independent and random process. Some sections may contain one or more target molecules, while others may contain none. Typically 20,000 sections are made and amplified. The cleavage is performed by microplate or droplet method containing channels. Each section is subjected to PCR amplification up to the endpoint. Amplified samples and non-amplified sections are counted separately. With the endpoint detection method, each reaction well gives a “yes” or “no” answer. Those containing amplified product and showing a fluorescent signal are identified as positive and numbered “1”. Those without amplified product and showing only a background signal are indicated as negative and are numbered “0”. The number obtained was defined as digital PCR because it is binary. Poisson statistical analysis allows direct determination of the absolute DNA concentration of the target in the initial sample without the need for subsequent references or standards.
The assay components used in the dPCR reaction consist of the master mix, primers, TaqMan probes and target DNA, as in qPCR.
It performs the analysis by splitting or dividing each sample into a large number of separate and parallel reactions. The master mix with the diluted sample is divided and distributed into individual PCR reactions. The distribution of molecules within the sections is an independent and random process. Some sections may contain one or more target molecules, while others may contain none. Typically 20,000 sections are made and amplified. The cleavage is performed by microplate or droplet method containing channels. Each section is subjected to PCR amplification up to the endpoint. Amplified samples and non-amplified sections are counted separately. With the endpoint detection method, each reaction well gives a “yes” or “no” answer. Those containing amplified product and showing a fluorescent signal are identified as positive and numbered “1”. Those without amplified product and showing only a background signal are indicated as negative and are numbered “0”. The number obtained was defined as digital PCR because it is binary. Poisson statistical analysis allows direct determination of the absolute DNA concentration of the target in the initial sample without the need for subsequent references or standards.
Workflow
– Reaction Preparation
– Partitioning Droplet Nanofluidic Chip
– Amplification Transfer
– Reading
– Transfer to Software
Digital PCR’s Progress by Year
– 1992 First dPCR system designed and described.
– 1999 The first paper using the term digital PCR is published.
– 2007 The first commercial dPCR system is introduced based on microfluidic chips and microarray.
– 2010 The first commercial dPCR system is introduced based on rotating microfluidic disks.
– 2011 First commercial dPCR system based on water-oil emulsion/droplets is introduced.
– 2017 First commercial microplate-based dPCR system introduced.
– 2020 First commercial nanoplate-based dPCR system launched.
qPCR Advantages According to
Quantitative PCR (qPCR) is a well-established and preferred method for relative quantitation in time-demanding routing applications, resulting in a wide dynamic range, high throughput and screening of large numbers of samples.
Given the advantages of dPCR, it has found many uses and this potential continues apace. Absolute quantitation offers significant advantages over qPCR, enabling reproducible measurements of low target DNA in complex backgrounds with high sensitivity.
It is a powerful platform that surpasses many other methods thanks to its potential to quantify very small amounts of target DNA in minutes.
It has also allowed dPCR to be improved due to the increased tolerance to inhibitors and amplification efficiency thanks to partitioning, or due to technical limitations in the calibration of standard curves generated in qPCR. All these features make dPCR a simple and cost-effective next generation technology.
qPCR Advantages
– Well-established protocol and data analysis techniques
– Large dynamic range
– Low cost per sample
– High sample throughput
dPCR’s Advantages
– No need for a standard curve or reference sample
– High tolerance to inhibitors
– Superior and increased sensitivity
– High reproducibility
– Simple workflow even for small samples
– Ability to increase sensitivity by using more PCR replicates
– Linear detection of small-fold changes
dPCR’s Disadvantages
– Not very widespread yet
– Lack of validated methods
– Lack of commercial kits
Whether the technology will mature and whether the cost will decrease is a matter of curiosity for many researchers. Although it has few applications at the moment, it is expected to have many more areas of use tomorrow.
The methods are complementary to each other. Therefore, dPCR will be able to provide a more confirmatory approach to those who want to achieve precision in their fields of use.
dPCR’s Areas of Use
Copy number variations;
– Rare Mutation Detection
~Detecting cancer below current detection levels
~Detecting novel mutations and duplications in cancer
~Monitoring rare drug resistant mutations
– Viral load detection
~Bacterial load
– Gene expression
~determination of mRNA levels
– Investigation of methylation events
– Quantitation of NSG libraries
– Absolute quantification (presence/absence analysis)
– Single point mutations (SNP)
– GMO analysis
– Genotyping
– Infectious diseases (FactorV, MTHFR etc.)
– Environmental testing
– Prenatal diabetic
– Organ transplants
– Determining the efficiency of DNA extraction
– Quantitation of low-level pathogens
– Being a reference method in proficiency tests
– Better comparison of results between laboratories
Why? To dPCR shall we?
The ability to obtain highly sensitive quantitative results without the need for a standard curve between nucleic acid targets seems to be the greatest strength of dPCR.
Cost-effective method for pathogen analysis and GMO detection.
High sensitivity in inhibited samples and seed samples.
Simplified result interpretation system.
Improved sensitivity in sub-optimal tests.