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Digital PCR

dPCR for beginners

Evolution of dPCR

Today's complex research questions demand a depth of information beyond the capacity of traditional PCR technologies. Third-generation digital PCR is reducing that gap and becoming a much simpler and more practical technique to address these everyday research questions.

The concept of digital PCR has been around since 1992 when Sykes et al. described it as "limiting dilution PCR." This general method used end-point analysis and Poisson statistics to quantify the absolute number of nucleic acid molecules present in a sample. What followed was the revolutionary work by Vogelstein and Kinzler in 1999, who developed a method whereby the sample was diluted and distributed into individual reactions called partitions, and single products with fluorescence signals were detected and analyzed after amplification. They then coined the term "digital PCR," as we all know it today.

Over the years, these methods have been innovated and commercialized for wider adoption. One can perform digital PCR on microfluidic chips and discs, microarrays and microdroplets or droplet crystals based on oil-water emulsions, and more recently, in qPCR-like plates.

Still looking for answers to what is digital PCR? Check out our bench guide to learn more about the fundamentals, advantages, limitations and applications of digital PCR.

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Learn what digital PCR can do for you

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Digital PCR enables absolute quantification of nucleic acids without the need for references or standard curves. The method, which partitions the sample into thousands of individual reactions, exhibits high tolerance to inhibitors, superior precision, increased sensitivity and high reproducibility. Due to these features, more and more researchers are applying digital PCR in their copy number variation analysis, rare mutation detection, viral load detection, gene expression analysis, next-generation sequencing library quantification and other applications.

Sample dilution and PCR reaction mix setup
Sample dilution and PCR reaction mix setup
Blue - Target Red - Background (gDNA, cDNA; primers/probes; master mix)
PCR reaction partitioning into thousands of individual reactions​
PCR reaction partitioning into thousands of individual reactions​
End-point PCR amplification of partitions
End-point PCR amplification of partitions
Green - Positive reactions Blue - Negative reactions
Readout and absolute quantification​
Readout and absolute quantification​
Divide and conquer

While the sample is prepared like that for qPCR, sample partitioning where a sample is divided into thousands of individual reactions before amplification is unique to digital PCR. By random distribution of molecules into partitions, unlike the bulk analysis in qPCR, digital PCR minimizes the effects of competing targets and enhances the precision and sensitivity power of detection of the rare.

It allows researchers to:

  • Quantify low-abundance targets or targets in complex backgrounds
  • Detect and discriminate allelic variants (SNPs)
  • Monitor small fold changes in target levels otherwise undetectable by qPCR
digital PCR, dPCR, infographic, downloadable, workflow, applications
Increases effective concentration​
  • Improves limit of detection (LOD)
  • Amplifies and detects single target molecules
Confers enrichment effect​
  • Increases the ratio – target vs. background
Provides superior precision and linearity​
  • Measures individual molecules vs. an ensemble concentration
  • The more the partitions the higher the precision
  • The more the partitions the broader the dynamic range
Choosing dPCR vs. qPCR
Compare and contrast the two methods to understand which one best suits your needs and learn how to transition your current qPCR assay to dPCR.
Poisson’s law gives meaning to partitioning

Contrary to real-time qPCR, digital PCR does not rely on every amplification cycle to determine the relative amount of target molecule; rather, it relies on Poisson statistics to determine the absolute target quantity following an end-point amplification.

As the target molecule is distributed randomly across all available partitions, Poisson distribution estimates the average number of molecules per partition (zero, one or more) and calculates the copies of the target molecule per positive partition. Poisson statistical analysis of the number of positive and negative reactions yields precise, absolute quantitation of the target sequence.

Applying the statistics to absolute quantification

In the context of a digital PCR experiment, absolute quantification relies on the random distribution of target molecules across the partitions and the data is expected to fit a Poisson distribution. Poisson distribution got its name in 1837 after the French mathematician Siméon Denis Poisson and is applied to the probability of a given number of events in a fixed period if the events occur at a known constant rate and are independent of the occurrence of the previous event.


Beginner’s guide to dPCR
We’re here to guide you through the workflow, with tutorials and planning your first digital PCR experiment.
Finding a needle in a haystack – the power of digital PCR​
Finding a needle in a haystack – the power of digital PCR​

Speaker: Laima Antanaviciute, Ph.D., Market Development Manager – dPCR/PCR, QIAGEN

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Advantages of digital PCR and its applications – the power of partitioning
Advantages of digital PCR and its applications – the power of partitioning
Speaker: Daniel Heinz Löfgren, M.Sc., Market Development Manager – dPCR/PCR, QIAGEN
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The use of digital PCR for high accuracy molecular measurement​
The use of digital PCR for high accuracy molecular measurement​
Speaker: Dr. Jim Huggett, Principal Scientist, National Measurement Laboratory
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What is the difference between absolute and relative quantification?
Absolute quantification using digital PCR simply counts the number of molecules present in a sample. No known standards are required. In absolute quantification using the standard curve method in qPCR, the unknowns are quantified using a known amount. Relative quantification uses a reference sample to compare and determine the number of original copies of template DNA in the reaction of interest relative to the reference sample.
What are the applications of PCR techniques?
The applications of PCR techniques include determining the presence or absence of a target nucleic acid, genotyping, quantifying gene expression, validating assays, detecting microbial targets, quantifying NGS libraries and others.
How accurate is dPCR?
Digital PCR is an accurate method for quantifying nucleic acids. Studies show dPCR substantially improves precision in resolving a small number of copies or counting single molecules, especially in the presence of inhibitors or wild-type populations. Digital PCR has lower detection limits and higher intra-lab reproducibility compared to traditional qPCR.
How does dPCR work?
You'll be relieved to know that the initial dPCR reaction is assembled using familiar assay components similar to qPCR. But then, by discretizing or partitioning each sample into a large number of individual and parallel reactions, you're left with one or more target molecules in some partitions, whereas others may contain none. Partitioning can be achieved by dividing the sample into microplates containing capillaries or channels, arrays of miniaturized chamber, or oil-water emulsions as droplets. Each partition undergoes PCR amplification to the endpoint. Partitions with and without amplified products are individually counted. Those containing amplified products and showing a fluorescent signal are designated as positive and scored as "1"; those with no amplified product and only a background signal are designated as negatives and scored as "0". Poisson statistical analysis is then applied to determine the absolute concentration of the target present in the initial sample without relying on references or standards.
Which applications can benefit from using dPCR?
Applications requiring the detection of small amounts of input nucleic acid or the finer resolution of target amounts, such as rare event detection, copy number variation analysis and gene expression analysis of the low-abundance transcripts, can significantly benefit from the partitioning effect of dPCR. Applications such as liquid biopsy, single-cell analysis, NGS library quantification, quantification of low viral and bacterial loads, analysis of gene editing events and GMO detection can also leverage the tremendous precision and high sensitivity that digital PCR offers compared to qPCR. For most researchers, dPCR represents a complementary approach to qPCR.
What type of templates can be used for dPCR?
You can use starting material ranging from genomic DNA isolated from blood, tissues, cells, tumors, cell culture, swabs and lavages, FFPE DNA (formalin-fixed paraffin-embedded DNA), cfDNA (circulating free DNA), ctDNA (circulating tumor DNA), bisulfite-treated DNA, environmental DNA, cDNA, bacterial DNA, viral DNA, plasmid DNA, library DNA, and synthetic DNA such as gBlocks and longmer oligonucleotides to total RNA, microRNA, lncRNA, viral RNA, bacterial RNA and in vitro transcribed RNA.
Is digital PCR the same as digital droplet PCR?
No, because digital PCR is an umbrella term for all digital PCR methods, not just digital droplet PCR (ddPCR). For example, ddPCR uses droplets as partitions for the PCR sample, whereas other digital PCR methods are based on microfluidic plates, microfluidic chips or other principles.
How is digital PCR different from normal PCR?
This depends on the definition of normal PCR. End-point PCR relies on gel electrophoresis for qualitative or semi-quantitative analysis of nucleic acids. Quantitative PCR (qPCR) can measure nucleic acid concentrations in real time based on standard curves. Digital PCR offers absolute quantification of nucleic acids by combining end-point PCR with partitioning and statistics.
What is digital PCR test?
A digital PCR test is a method for absolute quantification of nucleic acids. The PCR reactions is divided into thousands of partitions, where the target nucleic acid is amplified individually. Fluorescence probes or dyes are used to detect the presence or absence of products in each partition.
What is the difference between digital PCR and ddPCR?
The difference between digital PCR and droplet digital PCR (ddPCR) is in the partitioning approach. In nanoplate dPCR, the sample is divided into thousands of partitions on a microfluidic dPCR plate. In droplet digital PCR (ddPCR), the sample is divided into thousands to millions of droplets, which are then analyzed one-by-one on a droplet reader.
Digital PCR news
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