The Importance of Speed in PCR

The job of a thermocycler is to implement a set of temperature changes in a reaction volume. The biological requirement for PCR is to implement a set of temperature changes such as this:

Repeat 30 times:

1. Denature at 95 C for 30 seconds
2. Anneal at 64 C for 45 seconds
3. Extend at 73 C for 45 seconds

If you add up the times required, the biological requirements of this reaction take 50 minutes. However it also takes time for the PCR machine to transition between each set of temperatures, which is effectively wasted time. Here’s where the speed of the PCR machine comes in. If the PCR machine is capable of changing 2 C/s, then starting from 20 C the above reaction would take 66 minutes. However if it was only capable of changing 0.5 C/s, the above would take nearly twice as long: 114 minutes.

When we sat down to design a low cost thermocycler, we had options that were cheap but slow, such as relying on convection and radiation to cool the heat block. We opted instead to go with a more complex peltier cooling solution so the machine would be faster: our prototype currently ramps at about 2 C/s.

Why did we feel speed was so important? There’s two reasons.

My formal background is in computer science. When writing new software code, I do not write it correctly the first time. I may have a design flaw, or simply have made some mistakes. However I’m able to get rapid feedback, correct the issue, and test it again, so making mistakes is not a problem at all. In software, it’s typically possible to get feedback in a time ranging from seconds for small changes to at most about 10 minutes to run a well designed test suite on a modestly sized application. That rapid feedback cycle not only allows engineering projects to proceed rapidly, but it is also a large part of how the novice developer learns the art of software development.

When I work in biology, my experience is anything but that. Experiments (or design-build-test cycles) run anywhere from hours for simple operations, to days if cells must be grown, to weeks if DNA must be synthesized, to years for long-running clinical trials. Faster PCR cannot solve all of that, but it can do its part for the experiments where it is critical.

For example, I’ve been working to create a SNP genotyping protocol which would allow people to read any SNP of interest from their own DNA. It’s complex and involves DNA extraction, PCR, and a gel run. Currently it takes the better part of a day to complete; reducing it so it could be run twice a day would be a big win, especially considering someone new to biology is likely to make mistakes the first couple times. We should all be thinking about how we can make design-build-test cycles faster when we create tools for biology.

The second reason is a little more technical. The PCR reaction makes use of a “thermostable” polymerase enzyme, which really means that the polymerase doesn’t denature and cease to function right away at the denaturing temperatures of PCR. In actuality the polymerase has a half-life which is reduced as temperature increases. At the end of a 25 cycle PCR run, the majority of the polymerase may in fact be destroyed, which is a limiting factor in the amount of amplification possible.

That can be a problem if you’re starting from a very small amount of DNA, as may be the case in a forensics application. It is also important if your upstream DNA preparation protocol is sub-par. For example, suppose a DIYbio enthusiast was doing sushi DNA barcoding. While they have access to an enormous amount of DNA to begin with, they may use a lower-cost DNA extraction method which doesn’t remove as many PCR inhibitors as a Qiagen extraction kit would. That’s not a problem so long as you’re still able to get enough amplification for sequencing, which you can do by extending the number of PCR cycles so long as you have enough working polymerase left.

So faster PCR can introduce resilience to overcome poorer upstream processing, and if that enables you to get the same results with cheaper reagents, that’s another win for everyone.