Capable of detecting both TaqMan® and SYBR® chemistries (FAM™, VIC™, SYBR™, ROX™), dPCR offers absolute quantification and rare allele detection by directly counting the number of target molecules present in a sample, rather than relying on reference standard curves or endogenous controls, as used in qPCR. qPCR still maintains the advantages of enabling high throughput and a wide dynamic range for your assays, but dPCR should be considered to be a great compliment to qPCR. A simple example of this may be a case where you record a high Ct value on a qPCR run. You’re pushing in to the limits of detection of the assay and/or instrument, and the precision of the measurement is not adequate for you to call the final result. dPCR could be used here to achieve an accurate result, without the need for redesigning your experiment.
A key feature of dPCR is the method of measuring nucleic acid amounts. Rather than carrying out the PCR for a sample in a single reaction tube, in dPCR, the sample is spread or “partitioned” across a large number of discrete wells on a high-density nanofluidic chip. Each chip contains 20,000 wells (partitions) and the PCR is carried out in each partition. During the partitioning, some partitions will have received the nucleic acid sequences of interest, while others will not. At the end of the PCR, each partition acts as a data point, and as such, thousands of data points can be analyzed in a given run.
At this stage, it is important to point out the limiting features during a traditional PCR. During the exponential phase, there is exact doubling of product at every cycle, and this is possible because all reagents are fresh and available. Through the linear phase, the reactions start to slow down as reagents are consumed as a result of amplification and PCR product is no longer being doubled at each cycle. Finally, at the plateau phase, the reaction has stopped and no more products are being made.
So how can this affect the detection of a rare target?
Perhaps I have a multiplex reaction where I’m trying to amplify a rare mutant allele in a pool of wildtype alleles. If the wildtype alleles are present at a high level, which they most likely are, this can interfere with the detection of the mutant allele. Reaction components are getting used up before the mutant allele can be amplified to a signal that’s detectable.
However, thanks to the partitioning effect in dPCR, the amplification of one target doesn’t affect the amplification and subsequent detection of the other, since they are now partitioned separately. Essentially, you are able to enrich for the sequences of interest and dilute out the wildtype background.
Aside from this competitive effect, at low initial concentrations of target in a given sample, target nucleic acid molecules simply may not amplify to detectable levels in qPCR, whether that’s a single or multiplex set-up.
The dPCR Workflow
The first step is to set up the dPCR reaction by mixing sample, Digital PCR master mix and TaqMan® or SYBR® assay(s) to a final reaction volume of 14.5 mL per sample. ROX™ is included in the master mix to enable the instrument to differentiate between the wells that received reaction mix and those that didn’t, and this factor is taken in to account later during the analysis stage.
The dPCR reaction mix is partitioned across a QuantStudio® 3D Digital PCR Chip using an automated Quantstudio® 3D Digital PCR Chip Loader that also fixes a lid to the chip. This takes about 30 seconds. Each chip has a unique identifier code to help with sample tracking. The chip is filled with Immersion Fluid through a loading port on the lid, which is then sealed. The PCR is then performed using a flat-block conventional thermal cycler, e.g. - a ProFlex™ 2x Flat PCR System or Dual Flat Block GeneAmp™ PCR System 9700. Partitions with the target molecule will amplify during the PCR to a detectable level.
Once the PCR is completed to end-point, the chip is read on the QuantStudio® 3D Digital PCR Instrument. This takes about 30 seconds. Based on the detection of fluorescence, the instrument determines whether or not amplification occurred. Each reaction well/partition is characterized as positive or negative, and the number of positive reactions is directly proportional to the total number of molecules present in a sample.
However, some wells on the chip will receive more than one target molecule during the partitioning stage. The instrument can automatically correct for this effect by using standard statistics on the number positive and negative wells that are identified. A Poisson model is used to calculate the probability of a given reaction well receiving zero, one, two, three or more copies. This correction factor enables all molecules in the starting sample to be accounted for, and so an absolute quantification is established.
Because dPCR does not rely on Ct values to quantify copy number, comparison to a known standard is not required for absolute quantification. This demonstrates that dPCR is also useful to develop a reference for your real-time PCR experiment if one doesn’t exists, i.e. – for absolute quantification applications.
The results, given in “copies/mL”, can be viewed on the instrument touchscreen. For each answer calculated, a data quality assessment is made. Data considered to be of marginal or failing quality are then appropriately flagged for further review on the cloud-based QuantStudio® 3D AnalysisSuite™ Software.
While dPCR enables increased sensitivity and specificity of target detection thanks to the partitioning of samples in to individual reactions, thus reducing competition of background DNA, you can always enhance these factors further, i.e. - Increasing the volume sampled will increase the sensitivity, and increasing the number of chip replicates for a given sample increases the statistical significance of your answer. On the cloud-based software, data from multiple chips for a given sample can be combined.
The dPCR set-up takes just a few minutes and avoids some of the common problems associated with droplet PCR. The chip-based dPCR workflow is more streamlined with a lower amount of pipetting steps. It is also more secure in that you’re working with a sealed system once the chip is closed. In comparison, current droplet-based methods require multiple pipetting and transfer steps. This exposes the reaction mixture which may lead to cross-contamination of samples and/or contamination of your lab with rogue amplicons. The partitioning approach also avoids problems of amorphous droplets that are prone to coalescence and shearing.
dPCR also offers a lower cost-per-sample on a low sample throughput level.
It is also worth noting that the system is highly tolerant to inhibitors that may be present in a sample. As the target molecules are partitioned across the chip, so too are the inhibitors. Some partitions will have received inhibitors while others will not, meaning their impact on the reactions is reduced.
Applications for dPCR include rare cancer mutation quantification, copy number variation (CNV) analysis, pathogen detection and load determination, absolute quantification of standards, library quantification for next-generation sequencing, characterization of low-fold changes in mRNA and miRNA expression, GMO detection and contamination assessment.
To note, Custom TaqMan® SNP Genotyping Assays used to quantify the most common cancer-related mutations (e.g. - EGFR, BRAF, KRAS, PIK3CA, JAK2) are included in the QuantStudio® 3D rare mutation analysis solution. These assays were designed using the TaqMan® Assay design bioinformatics pipeline and then validated specifically for the QuantStudio® 3D Digital PCR System. Combining TaqMan® fluorogenic 5’ nuclease chemistry with digital PCR methodology, researchers are now able to detect and quantify rare mutants at a prevalence level as low as 0.1%.
The The QuantStudio® 3D Digital PCR System – a simple and robust operation with a sealed chip design, bringing high-performance quantification to your lab.
David O’Neill, PhD
Field Application Specialist