Digital PCR (dPCR) is a nucleic acid quantification technology which enables users to count the absolute number of target nucleic acid molecules in a sample without the need for a standard curve. To do this, a bulk PCR sample with nucleic acid and assay is separated or partitioned into a large number of small, isolated PCR reactions. Although a variety of methods have been developed and commercialized, each suffers from limitations that prevent users from reaping the full benefits of dPCR.
At its introduction in 1988, a 384-well plate was used to demonstrate the principles of “limiting dilution” for rare target detection1. As dPCR technology have progressed, so have methods of reagent partitioning. One of the most common methods available today is the use of microfluidic technology to create many small, isolated reactions. This enables tens of thousands rather than hundreds of much smaller volume partitions to be generated per dPCR reaction. Increasing the number of partitions improves the ability to detect rare targets as wells as improves accuracy for higher concentration targets – essentially expanding the dynamic range2,3.
Although powerful, droplet technology suffers from a lack of consistency due to the dependency on an inherently stochastic process – fluid shearing – to create individual partitions. Therefore, the total number of partitions generated per dPCR reaction using these methods can be highly variable. Microfluidic Array Partitioning (MAP) technology, on the other hand, does not rely on fluidic shearing used in droplets or a physical displacement of excess reagents to form partitions. This innovation provides superior consistency, reduces reagent waste, and a simplified workflow. We will be focusing on the improved consistency MAP provides by covering consistency in total acceptable partitions, overall volume precision, and quantification.
So what does Microfluidic Array Partitioning technology look like? The MAP plate (shown below) has 16 dPCR reaction units, easily distinguished as opaque squares. Zooming in, each dPCR reaction unit (opaque square) is made up of 20,480 fixed array partitions. The partitions themselves are connected to a central channel which is used to deliver PCR reagents from a single upstream well. With a 4-column and 4-row design, as few as four and as many as 16 dPCR reactions may be run in parallel per plate. In this article, we will be focusing on consistency in total acceptable partitions across MAP plates.
We conducted a study testing the limit of detection for 5 separate oncology assays, and in parallel selected this data set to demonstrate the repeatability of high accepted partition count across the MAP plate for different assays. After dPCR was complete, our |Q| Analysis software finds and inspects each partition within a unit using a QC channel to identify only those partitions which are acceptable for analysis – removing obvious debris such as dust or or partitions appear non-uniformly filled. For this study, 2 separate channels were subsequently used for detection of wild-type (VIC) and mutation (FAM) signal.
Across the five plates included in the study, we identified an average partition acceptance of 99.9% (±0.5%) of the total partitions (partitions accepted / 20480 total possible). We then plotted the total number of accepted partitions per unit across each of the five plates below to show the overall consistency across each of the plates. On the plot are two dashed lines. The first at 20,000 indicates the ideal targeted partition count for current dPCR platforms. The second at 20,480 indicates the total number of partitions available to fill on the MAP plate. With the exception of 1 unit out of the 80 units tested, all dPCR reactions generated >20,300 accepted partitions – well above the typical target.
So why does the number of partitions analyzed matter? The answer is rooted in statistics!
Using Poisson modeling (above) both the total number of partitions analyzed and the partition volume are used to calculate final concentrations from dPCR reactions. So it is critical for both of these variables to be highly consistent and calculated precisely. Microfluidic Array Partitioning technology improves consistency in total acceptable partitions, overall volume precision, and as a result overall quantification.
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- Saiki RK, Gelfand DH, Stoffel S, et al. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science. 1988;239(4839):487‐491. doi:10.1126/science.2448875
- Huggett JF, Foy CA, Benes V, et al. The digital MIQE guidelines: Minimum Information for Publication of Quantitative Digital PCR Experiments. Clin Chem. 2013;59(6):892‐902. doi:10.1373/clinchem.2013.206375
- Quan PL, Sauzade M, Brouzes E. dPCR: A Technology Review. Sensors (Basel). 2018;18(4):1271. Published 2018 Apr 20. doi:10.3390/s18041271