What is digital PCR and where did it come from?
Digital PCR (dPCR) is an emergent technology that is poised to become the new gold standard in precise nucleic acid quantitation. However, it is not a brand new concept. Digital PCR, or limiting dilution PCR as it was first described, works by dividing a PCR reaction mixture into a large number of small, individual reactions. This feature enables it to overcome many of the limitations of quantitative PCR (qPCR), such as need for standard curves, inability to accurately quantify low number of target molecules and lack of sensitivity in high background conditions.
So, how does it work?
If you have tuned into our webinars, you may be familiar with our very simplified description of the concept of digital PCR as a strategy for playing “Where’s Waldo?”.
In a sea of people and background events, Waldo is extremely challenging to find. However, if you break the scene up into discrete sections, you can focus on individual parts of the picture rather than the scene as a whole. If you gave each person in the isolated sections a red card and a green card, and asked all non-waldos to hold up red cards and all waldos to hold up green cards – the game is just counting!
This is essentially how digital PCR works. We split up a PCR mixture among tens of thousands of individual partitions, send in probes specific for our target of interest, then count how many targets were present. Since partitioning is the key to digital PCR, when comparing different digital PCR methods, it’s important to look at partition volume and number.
Partition Volume: This is used to calculate the concentration of your target. It’s important to know how much of your sample is being analyzed.
Partition number: The total number of pieces your sample is being divided into. More partitions allow for precise and accurate quantification of your targets.
How are Combinati’s digital PCR and droplet digital PCR methods different?
Most digital PCR technologies can be distinguished based on how reagents are partitioned as this can be achieved in a number of different ways. One of the most common methods seen today is droplet digital PCR or ddPCR. Unfortunately, generating droplets is a stochastic process, which can lead to unintended variability in droplet size and total droplet count depending on the assay, run, or technical skill of the user.
Combinati’s team designed a micro-molded plastic plate for digital PCR using microfluidic array partitioning (MAP) technology to create 20,000 precise partitions. Using Combinati’s platform, 9µL of PCR mix is loaded into the MAP16 plate and you can expect > 95% of our 20,480 fixed partitions to be filled with your sample. You can read more about the performance, here. In comparison, if you load 20µL of PCR mix onto a ddPCR platform and only obtain 10,000-15,000 droplets, you are throwing away a large portion of your PCR mix. This can be extremely problematic for both extremely rare samples (throwing away most of your sample) or for samples which are high in concentration (fill too many of your partitions and throw off Poisson statistics).
Thinking back to Waldo, this functionally means that analyzing more partitions and more sample volume enables you to “see” more of the picture, resulting in more accurate results.
Some existing digital PCR platforms can have 30-75% dead volume, which means that the remaining portion is un-analyzed. In the figure below, this is represented as portions of the picture being removed. As you can see, since this would happen randomly, in some cases you may never find Waldo.
Because of Combinati’s proprietary microfluidic technology and fixed volume partitions that hold a precise volume, the Absolute Q has a dead volume of approximately 5%, so 95% of your sample is used in the digital PCR reaction. A high total analyzed sample volume, paired with a consistent 20,000 partitions per reaction, makes the Absolute Q the most accurate digital PCR platform.