Real-time PCR has become an increasingly useful and popular tool in the lab, but with machines costing tens of thousands of dollars, it’s use has been out of reach to those without big budgets or the ability to leverage a well-equipped wet-lab. If you’re a biohacker you can basically kiss the thought of using real-time PCR, or qPCR, goodbye. Right? Well, not for long. SynBioBeta recently interviewed Josh Perfetto of Chai Biotechnologies to learn more about the Open qPCR (@openqpcr) Kickstarter Project.
What is Chai Biotechnologies?
Chai was founded in late 2013 to make the basic tools of molecular and synthetic biology more accessible — meaning not only cheaper, but also better designed, easier to use, and more open.
Where did Open qPCR come from?
After creating the earlier OpenPCR, I was working on a synthetic biology project, and needed to quantify gene expression. So I picked up a clunky 10 year old Real-Time thermocycler for about $5,000 at auction. It was terrible – I literally spent days trying to get the parallel port to work with a modern computer.
But what happened next surprised me — I fell in love with the machine and its capabilities, not just for quantification, but for a whole assortment of advantages. Now that I had the machine, I used it for almost every PCR reaction I ran. I realized so many colleagues hadn’t realized these advantages mainly because they had never had access to such a machine — they were too expensive unless you truly needed their quantification capabilities. Thus, the idea for Open qPCR was born.
Who can use Open qPCR?
It’s targeted pretty broadly: certainly any lab without access to a Real-Time thermocycler, but also high school classrooms, and DIYbio groups and community labs.
What is Real-Time PCR and what is it used for?
Real-Time PCR, also frequently called qPCR, is a system that monitors the amount of amplified DNA present throughout a PCR reaction, and thus in “real-time”. The most obvious application is quantification of the original template, which is useful to measure gene expression.
But there are many more uses. For example we all know PCR primers can mis-prime, so a simple primer pair often doesn’t make a good diagnostic. But by adding a third oligo known as a probe, the detection can be made much more specific and almost immune to mis-priming.
But my favorite use is the most mundane. Real-Time PCR gives you immediate feedback on the outcome and quality of each reaction you run, including whether non-specific products are present. If something goes wrong, you will know it right away, without the need to run a gel. It’s a small thing, but there’s a lot of small things that can go wrong when you’re engineering a biological system. Having smarter tools at every step of the way enables us to focus on the bigger picture.
What’s the difference between Open qPCR and Open PCR?
There’s really no relation other than my personal history with both projects. OpenPCR was an endpoint PCR thermocycler sold as a build-it-yourself kit. Open qPCR is a Real-Time thermocycler sold as a professionally manufactured product. Open qPCR also has immensely better performance and software.
What are the specs of Open qPCR?
It has 16 200 uL reaction wells, temperature ramps of 5 C/s, Real-Time detection of green fluorophores, a modern web interface, touchscreen, and ethernet, wifi, and USB interfaces.
In addition to the open-source hardware of Open qPCR, the system comes with open-source software for visualizing each run. What’s possible with the new software program?
The software supports amplification curves, presence/absence detection, melt curves, and we’re currently implementing relative quantification. It also has a very modern, well-designed web interface, including a visual protocol and plate editor, which makes the machine very easy to use.
Typical qPCR machines cost $20,000 or more. What allows Open PCR to offer Open qPCR for a fraction of that cost?
We make use of open source software and hardware such as the Beaglebone Black controller board, and also limited the machine to a 16 well block and single fluorophore detection. These certainly will be unacceptable limitations for some, but it makes the device affordable to a multitude of users who don’t need more advanced systems.
What types of reagents can I use with Open qPCR?
You can use any dye/probe with a green fluorophore like FAM. We’ll also be releasing our own IP-free intercalating dye known as Chai Green.
Good news for all
The project reached its funding goal of $50,000 pretty quickly and has since climbed to over $134,000, which means Open qPCR will be available for everyone long after the Kickstarter Project ends. And while the funding goal was reached, you can still purchase a low-cost, high-quality Open qPCR machine today by visiting the project page.