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.
Introduction to dPCR – Learn how the technology works and what it can do for you
The power of digital PCR
Digital PCR is a highly precise approach to sensitive and reproducible nucleic acid detection and quantification. Measurements are performed by dividing the sample into partitions, such that there are either zero or one or more target molecules present in any individual reaction. Each partition is analyzed after end-point PCR cycling for the presence (positive reaction) or absence (negative reaction) of a fluorescent signal, and the absolute number of molecules present in the sample is calculated. It does not rely on a standard curve for sample target quantification. Eliminating the reliance on standard curves reduces error and improves precision.
Absolute quantification in 4 steps
Sample dilution and PCR reaction mix setupBlue - Target
Red - Background (gDNA, cDNA; primers/probes; master mix)
PCR reaction partitioning into thousands of individual reactions
End-point PCR amplification of partitionsGreen - Positive reactions
Blue - Negative reactions
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
Top 3 advantages of partitioning
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
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.
Finding a needle in a haystack – the power of digital PCR
Speaker: Laima Antanaviciute,
Ph.D., Market Development Manager – dPCR/PCR, QIAGEN
Advantages of digital PCR and its applications – the power of partitioningSpeaker: Daniel Heinz Löfgren,
M.Sc., Market Development Manager – dPCR/PCR, QIAGEN
The use of digital PCR for high accuracy molecular measurementSpeaker: Dr. Jim Huggett,
Principal Scientist, National Measurement Laboratory
Quick answers to FAQs about digital PCR
- Vogelstein B and Kinzler KW (1999). Digital PCR. Proc Nat Acd Sci USA 96, 9236–9241.
- Baker M (2012). Digital PCR hits its stride. Nat Methods 9, 541–544.
- Pohl G and Shih IeM (2004). Principle and applications of digital PCR. Expert Mol Rev Diagn 4, 41–47.
- Sykes PJ et al. (1992). Quantitation of targets for PCR by use of limiting dilution. BioTechniques 13, 444–449.
- Morley AA (2014). Digital PCR: A brief history. Biomol Detect Quantif. 1(1):1-2.
- Quan PL et al. (2018). dPCR: A Technology Review. Sensors (Basel). 18(4):1271.