What is ddPCR?

ddPCR (droplet digital PCR) is a breakthrough technology, a potential game changer in

the fields of life science and clinical diagnostics
_ It is a method that uses droplet technology to divide complex samples into small,
manageable sub-units
_This technology is (so far) used for DNA and RNA, but should soon expand to other
targets
_ It provides absolute and extremely high resolution for target quantification

Benefits of ddPCR

Absolute Quantification: No need for a standard curve because number of targets in a

given volume (i.e. concentration) is counted directly (and adjusted for multiple
targets/droplet by Poisson statistics)
• Precision: The ability to consistently make the same measurement on replicates of a
sample (minimal CV across msmts)
• Reproducibility: The ability of a researcher to obtain the same measurement on the
same sample from experiment to experiment in the same lab OR in another lab across
the globe.
• Sensitivity: The ability to measure very few copies of a target with precision mostly
limited by the technology independent statistics of sampling
• Accuracy: The ability to make the correct measurement – this requires validation
before this can be claimed, but is then an intrinsic property of the ddPCR assay used.

Droplet Digital PCR Workflow

Partition reagents and sample into thousands of droplets

_ Perform PCR on thermal cycler
_ Count droplets with a positive PCR product (fluorescent) and a negative PCR
product
_ Digital readout provides concentration of target DNA

Droplet Fluorescence Converted to a Digital Signal

Positive droplets contain at least one copy of target DNA (cDNA)

_ Positive droplets have increased fluorescence vs. negatives
_ Quantasoft software measures the number of positive and negative droplets per
fluorophore per sample

Rare Event Detection

ddPCR is very well suited for studies with limited number of targets, where sensitivity is
of the essence. The robustness of this method to PCR inhibitors is also an asset for
detection of rare targets in complex matrices (blood, plasma, environmental samples).
Gene Expression Analysis
Analysis of RNA (transcripts) levels in biological samples
_Usually performed in duplex (in combination with reference gene)
_ Includes detection of miRNA
_Growing research on single cell transcriptome

ddPCR is very well suited for studies with limited number of targets, where changes in
levels of expression are <2-fold. It is also useful for studies of alternate species (RNA

editing,allelic expression), in single cell studies or for low abundance targets .

Copy number and variation
tAnalysis of the change in ploidy of certain genes, genomic regions or chromosomes
• Can be associated with normal developmental processes or pathological evolution

• Important field of study for cancer, human genetics, crop studies… o

ddPCR is very well suited for studies with limited number of targets, where sensitivity
is of the essence (5 vs. 6 copies). It also allows the researcher to obtain information on
the physical linkage of the studied targets (is it a tandem amplification or an additional
chromosome?)

Best Reasons to use ddPCR

Sensitivity
–Detect rare mutations in complex backgrounds
–Detect rare mutations earlier
–10-1000x fold improvement over qPCR
–Works with FFPE samples
–Works with blood, tissues, environmental samples…
Absolute Quantification
–Answers in absolute numbers of molecules (not Cq)
–Quantify lower levels of targets
–No standard curve
Precision
–Measure more subtle differences in expression or mutation
–Detect structural variants in cancers

–Higher tolerance to PCR inhibitorsBest Reasons to