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.

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.

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

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.