Compartmentalization is the basis for digital PCR
 

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At its introduction in 1988, a 384-well plate was used to demonstrate the principles of “limiting dilution” for rare target detection (1). As DNA amplification and quantification using digital PCR (dPCR) has evolved, so have methods of reagent distribution or compartmentalization. One of the most common methods available today is the use of microfluidic technology to create many small, isolated reactions via emulsification into droplets. This enables tens of thousands of now much-smaller-volume droplets to be generated per dPCR reaction. Increasing the number of droplets improves the ability to detect rare targets and improves accuracy for higher concentration targets and essentially expanding the dynamic range (2,3).


Precise and accurate digital PCR
 

The Applied Biosystems QuantStudio Absolute Q Digital PCR System relies on its proprietary microfluidic array plate (MAP) technology to deliver highly-accurate dPCR results. Here’s how it works:

The MAP (Figure 1) 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 microchambers. The microchambers themselves are connected by a distribution network that is used to deliver PCR reagents. Once the reagents have been compartmentalized into the microchambers, PCR amplification then proceeds, and the number of microchambers with successful DNA amplification are counted.

Microfluidic array plate graphic
Figure 1. Microfluidic array plate technology


Greater consistency than other dPCR methods
 

Some emulsion-based or droplet digital PCR (ddPCR) technologies suffer from a lack of consistency due to the dependency on an inherently stochastic process—fluid shearing—to create individual compartments. This can result in the total number of compartments generated per dPCR reaction being highly variable.

QuantStudio Absolute Q MAP technology, on the other hand, does not rely on fluidic shearing used in droplets or a physical displacement of excess reagents to form compartments or microchambers. So, in addition to delivering superior consistency, reduced reagent waste, and a simplified workflow, MAP technology provides overall greater volume precision, higher numbers of microchambers generated, and more accurate quantification.


A look at the data
 

To demonstrate the type of data generated by the MAP16 plate on the QuantStudio Absolute Q system, we used a multiplex assay that uses all four optical channels of the QuantStudio Absolute Q system to detect Target 1 (FAM), Target 2 (VIC), Target 3 (ABY), and Target 4 (CY5). Figure 2 shows the resulting dPCR fluorescence plots and maps of microchamber positivity generated by the QuantStudio Absolute Q Analysis software from a single reaction on the MAP16 plate.

Data MAP 16 plate
Figure 2. MAP16 plate showing multiplexed run using 4 channels.


Generate 20,000 dPCR microreactions—every time
 

To highlight the exceptional consistency of microchamber filling, we conducted a study to demonstrate the repeatability of high accepted microchamber counts across the MAP for different assays (Figure 3). For this study we ran 14 plates using in-house standard QC assays. In the dPCR workflow, once dPCR is complete, QuantStudio Absolute Q Analysis software finds and inspects each microchamber within a dPCR reaction or array using a QC channel, then accepts only those microchambers with uniform fill and no obvious signs of debris of non-PCR-related auto-fluorescence. Across the 14 plates included in the study, we identified an average microchamber acceptance of 99.7% (±0.6%). We plotted the total number of accepted microchambers per array across each of the 14 MAPs to show the overall consistency across each of the plates. The dashed line at 20,480 indicates the total number of microchambers available on the MAP. Across all arrays we noted an average of 20,417 (±133) microchambers accepted per plate.

Why does the number of microchambers analyzed matter?

Because statistics are at the root of dPCR analysis.

Using Poisson modeling (above), both the total number of microchambers analyzed and the microchamber 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. MAP technology improves consistency in total acceptable microchambers, overall volume precision, and, as a result, overall quantification.


References
 

  1. 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.
  2. 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.
  3. Quan PL, Sauzade M, Brouzes E. dPCR: A Technology Review. Sensors (Basel). 2018;18(4):1271.


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