Microfluidic Array Partitioning: Consistency in total partition count

Partitioning is the basis for digital PCR

Digital PCR (dPCR) is a PCR amplification technology which enables nucleic acid quantification by allowing users to count the absolute number of target nucleic acid molecules in a sample without the need for a standard curve. 

To accomplish this, a standard bulk PCR mix with nucleic acid assay is separated or partitioned into a large number of small, isolated PCR reactions. PCR amplification then proceeds within each isolated reaction, resulting in DNA amplification only if a copy of the target was originally present. Then, after amplification is complete, partitions which generated a positive PCR signal are counted – generating a simple absolute quantification. 

Although a variety of methods have been developed and commercialized, each suffers from limitations that prevent users from reaping the full benefits of dPCR.

A Breif History of digital PCR technologies

At its introduction in 1988, a 384-well plate was used to demonstrate the principles of “limiting dilution” for rare target detection1. As DNA amplification and quantification using dPCR has evolved, 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 via emulsions. This enables tens of thousands of now 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 range (2,3).

Droplet Digital PCR has limitations in consistency

Although powerful, emulsion-based or droplet digital PCR (ddPCR) 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. In addition to superior consistency, reduced reagent waste, and a simplified workflow MAP technology provides overall greater volume precision, higher numbers of partitions generated and more accurate quantification.

Microfluidic Array Partitioning improves consistency, maximizes sample utilization and simplifies workflow

 

So how does Microfluidic Array Partitioning technology work? 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 by a central channel which is used to deliver PCR reagents from a single upstream well. Once the PCR reagents have been loaded into the upstream well, Absolute Q applies positive pressure to the plate and reagents fill the fixed partitions. Next, Isolation buffer flushes the central channel – isolating or digitizing the reaction. PCR amplification then proceeds and the number of partitions with successful DNA amplification are counted. 

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.

Microfluidic Array Partitioning plates for digital PCR

To demonstrate the type of data generated by the MAP16 plate on the Absolute Q, here we highlight our novel multiplex digital PCR assay that measures four genes related to outcome for spinal muscular atrophy. This assay uses all optical 4 channels of Absolute Q to detect SMN1 (FAM), SMN2 (VIC), total SMN (TYE) and  RPPH1 (TAMRA) which is used as as reference. gene for copy number determination.  The figure below shows the resulting dPCR fluorescence plots and maps of partition positivity generated by the Absolute Q Analysis software from a single reaction on the MAP16 plate.

Generate 20,000 dPCR partitions - every time

To highlight the exceptional consistency of partition filling, we conducted a study to demonstrate the repeatability of high accepted partition count across the MAP plate for different assays. For this study we selected ran 14 plates using in house standard QC assays.  After dPCR is complete Absolute Q Analysis  both finds and inspects each partition within a unit using a QC channel, then accepts only those partitions 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 partition acceptance of 99.7% (±0.6%) of the total partitions (partitions accepted / 20480 total possible). We then plotted the total number of accepted partitions per unit across each of the 14 plates below to show the overall consistency across each of the plates. The dashed line at 20,480 indicates the total number of partitions available to fill on the MAP plate. Across all units we noted an average of 20417 (±133) partitions accepted per plate.

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. Interested in learning more about Microfluidic Array Partitioning technology? Sign up for our email list in the footer below to receive updates about new applications, events, webinars, publications, and more. If you would like to speak with an application scientist, get in touch by sending an email to info@combinati.com. We love to talk about digital PCR and see how the Absolute Q can fit your research needs!


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. doi:10.1126/science.2448875
  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. doi:10.1373/clinchem.2013.206375
  3. Quan PL, Sauzade M, Brouzes E. dPCR: A Technology Review. Sensors (Basel). 2018;18(4):1271. Published 2018 Apr 20. doi:10.3390/s18041271

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