Close

Does this project spark your interest?

Become a member to follow this project and don't miss any updates

$5 Polymerase Chain Reactor

The one of the most revolutionary inventions of the 20th Century, designed for DIY gene replication. (Now with a working prototype!)

13 103 65
Enjoy this project?
Share on twitter   Share on Facebook

This project was created on 07/11/2014 and last updated 10 days ago.

Description
The PCR machine allows us to copy, alter and join DNA in many ways, and has brought about the Biotech revolution. Until now, commercial PCR machines have cost thousands of dollars. OpenPCR has brought the price down to a few hundred, but that's still too high for classrooms, DIY'ers and HackerSpaces. But with this project, I aim to bring the price for a PCR machine down over two orders of magnitude, with a target price of about $5.

This project describes a remarkably simple but fully functional DNA replicator, based around the Arduino platform, and utilising just a handful of components. The trick is to use multiple physical process driven by just heat! Heat drives the conversion of DNA between its single and double stranded states; alters the activity of the polymerase in copying DNA; and drives circulation of the reaction mixture though several temperature zones via convection. Open Hardware. Open Software. Let's bring on the Biohacking Revolution!
Details

What's all this for then?

The Biotechnology Age is well under way. But like the Silicon Age, I think it will only really explode in growth once the entry price reaches a point where a few college dropouts can afford to build a biotech lab in their parent's basement, and with their inventions, put a dent in the universe.

The biotech laboratory is getting more and more streamlined, and experiments that would once have taken weeks or months can be performed in days, with much improved Recombinant DNA techniques, next-day-delivery of DNA primers, and cheap Gene synthesis. Although we can now forget about many old fashioned (but still widely used) techniques like Plasmid Cloning by Restriction Enzyme Digest, which require boxes full of expensive enzymes, the one technique that kickstarted the Biotech Revolution is still an absolute necessity.

PCR, or the Polymerase Chain Reaction, is one of the greatest inventions of the 20th Century. With it we can take a single stand of DNA and make trillions of identical copies. With these copies, we can identify a murderer from a speck of skin, DNA barcode living creatures to study entire ecological systems or identify the cause of a disease for personalised medicine. And because at the fundamental level all life on Earth operates on the same basis, we can even swap the "code" of life between species, rewriting the schematics of living organisms.

The grass roots interest in DIY biotechnology is growing, with projects like Biobrick and the Glowing Plant Project making headlines around the world. But the fact is, it's still too expensive for most people to get involved. With a target price of $5 for the parts to build a working PCR machine, and a secondary goal of $50 to build a really great machine, I hope to change that. At that price level, everything changes. Suddenly schools can afford to do recombinant DNA experiments in science class, just as the Apple IIe changed my school experience. Poorer countries can use the same equipment for medical diagnosis, and BioMaker's can start working on those inventions that will make the world a better place.

Download the designs, build the Polymerase Chain Reactor, and Viva la Revolucion Biotec!

System Design Documents

Here is the overview for the cheap, and ridiculously cheap PCR machines.

The $5 PCR machine

  • minimal parts
  • V-USB interface to calibrate temperatures, and log raw data

The $50 full featured PCR Machine

  • Bluetooth interface
  • Cloud-storage of optimal Annealing temperatures and DNA/Primer trading-portal for DIYBIO
  • GUI control of PCR machine, Primer annealing temperature calculator, Cloud access for sharing data
  • Realtime plotting of temperature data
  • I2C temperature sensors for ±0.4 Celcius accuracy, with no calibration required
  • Temperature fuses for safety
  • RGB LED for temperature status/bluetooth connectivity
  • Touchdown PCR and controlled annealing temperatures 

How PCR works:

In detail, the Polymerase Chain Reaction Machines operates by cycling a mixture of Template DNA (this contains the stuff we want to copy), Primers (short and cheap bits of DNA the match the front and back of the sequence we are copying), a Polymerase (the bio-nanobot that does the copying), and raw chemical nucleotides that will make up the sequence of the copies.

As an analogy, think of the template as a book, the primers as a set of bookmarks for certain pages, and the polymerase as a photocopier. The raw nucleotides are then something like the blank paper.  

PCR exploits the fact that some polymerases in Extremophiles from volcanic vents or hot springs can operate at very high temperatures, at which most organisms would be well and truly cooked.

Continuing with the (now very bad) analogy, imagine heating up the template as opening the book. Cooling the book inserts bookmarks at the correct location and closes it, and now you can hand it to the guy on the photocopier, who will photocopy everything between the bookmarks....

Read more »

Components
  • 1 × Arduino Nano
  • 1 × LM7805 Power Management ICs / Linear Voltage Regulators and LDOs
  • 2 × FQP2N60C Discrete Semiconductors / Power Transistors and MOSFETs
  • 2 × SBCHE1110RJ Resistors (Fixed) / Wirewound
  • 2 × LM335AZ/NOPB Sensors / Temperature, Thermal
  • 1 × perf board
  • 6 × Caps and resistors

Project logs
  • Building the Prototypes

    10 days ago • 0 comments

    Well, its been awhile since my last update, but that's not because of the huge amount of works I've been doing. I've been on holiday, but now I'm back working of the project

    Yay! I'm in the Top 50!

    Wow, I wasn't sure I would get this far, but it seems like I've passed the first barrier! I understand that the top fifty receive $1000 worth of electronic parts. Well, I hope they arrive soon and I can use them in this project, because I'm seriously low on cash to buy parts. I also hope they are sent from within Europe, because custom and duty fees are a killer!

    Anyway, I'm working hard on finishing working prototypes of the $5 board and the no-hold-barred super PCR machine. They are both really really really hard work, because at this stage, I don't have the time or funds to get PCB's made up. So, I am soldering all the parts by hand. And SC-70 surface mount parts are TINY!

    The only way I can manage it to to super glue the tiny temperature sensor to the aluminium heat spreaders upside-down, and then try and solder fine wire onto the legs, dead-bug style. Yes, I have destroyed many sensors so far....

    The heat spreaders are then held in place on the heating resistors with heating glue. 

    More to come soon!

  • Case Design and a New Direction

    a month ago • 0 comments

    I have started Sketching Up a few designs for the case, which I will have 3D printed soon. I plan on a compact case with a hinge design to allow for variable flow rates as I described in my last project log. This improved design marks the split from the $5 machine. I will finish designing the $5 perfboard-based machine, and post the code and specs, and will then focus on this much better version. The $5 machine is great for the classroom and people who want to try PCR out, but the new machine will be a practical machine for general purpose DNA replication.

    Features will include:

    • Low price
    • 3D printed case
    • Bluetooth connectivity
    • Cloud-Sharing of PCR parameters
    • Software interface
    • Touch-Down PCR capabilities
    • Heat-fuses for safety
    • I2C sensors for improved accuracy
    • Open source hardware and software

  • Huge success!

    a month ago • 0 comments

    Well, I had a theory that by tilting the orientation of the plane of the heating elements, I could adjust the flow rate. How would that work? Well, the convection current is caused by the different densities in the PCR reaction mixture at different temperatures. When the system is aligned vertically, there is the largest gravitational potential energy for driving the fluid flow. When the entire PCR tube setup is tilted, the vertical distance between the hotter and cooler fluids is reduced, so there is less drive pushing the fluid flow.

    Testing the theory, I tried amplifying a 1500 base pair sequence at three different angles, 90°, 60° and 30°. I have never had successful PCR reactions over 700 base pairs yet, and I haven't read of any successful reaction on any other convection-based machines either. 

    The PCR Machine was carefully adjusted to the correct angle using the complex apparatus shown in the picture below :) 

    Here is the Gel image of my three reactions:

    The top band in all three samples, at about 4500 base pairs, is the template DNA. But in the third PCR, tilted to 30° off horizontal, I have the right PCR product band! I'm incredibly excited, because it this means this isn't just a PCR machine for classrooms to demonstrate PCR anymore. When you can amplify products this size, you really have a great general purpose laboratory PCR machine that you can do real work on.

    That's not to say the earlier version wasn't useful. Diagnosing HIV, or doing DNA barcoding is possible with a machine that can only synthesis 700 base pair long products, but this new result open many many more possibilities.

    The next step with be to add a third heating element along the open side of the tube, to control the annealing temperature accurately. I will also have to add a mechanism that allows you to tilt the PCR heater easily, and maybe add dial-like markings to set the PCR length (i.e. 45° for a 1000bp long sequence). I might also replace the analog temperature sensors with I2C digital sensors, as this eliminates calibration issues with both the sensor itself and the ADC on the Arduino and only adds about 10 cents to the price of each heating element (when you buy hundreds or thousands).

    The project is moving along really well now, so please take the time to vote for the Polymerase Chain Reactor if you like it!

View all 11 project logs

Discussions

Jasmine wrote a month ago null point

Hello David, your project info is looking good, but please review your documentation to ensure it has everything we require for it to be considered for the next round of The Hackaday Prize.

By August 20th you must have the following info on your project page:
- A video. It should be less than 2 minutes long describing your project. Put it on YouTube (or Youku), and add a link to it on your project page. This is done by editing your project (edit link is at the top of your project page) and adding it as an "External Link"
- At least 4 Project Logs
- A system design document. Please highlight it in the project details so we can find it easily.
- Links to code repositories, and remember to mention any licenses or permissions needed for your project. For example, if you are using software libraries you need to document that information in the details.

You should also try to highlight how your project is 'Connected' and 'Open' in the details and video.

There are a couple of tutorial video's with more info here: http://hackaday.com/2014/07/26/4-minutes-to-entry/

Good luck!

Are you sure? [yes] / [no]

daveatfernie wrote a month ago null point

Progress certainly has moved on but now resembles the LavaAmp PCR machine :)

http://lavaamp.wordpress.com/

Are you sure? [yes] / [no]

David wrote a month ago 2 points

Interesting! The principle seems very similar, but that's not surprising. Once you start thinking about convection for PCR, the idea for a closed loop system becomes obvious :) Phil Howard came up with a similar idea, outlined in his comment below.

After a quick look though, a few points jump out:
- The circular design of the heaters is much harder to fabricate for DIY'ers.
- Attaching the tube to the outside of the heating element means the tube has to be a precise length to be in good contact (with my design, the tube presses outwards onto the heaters, and slack is used up in the corners, i.e. 5mm here or there isn't an issue.)
- The straight sides means I can use very cheap power resistors as heating elements. I guess by using wider aluminium heat spreaders, I could also PCR multiple samples like the LavaAmp, but thats not my goal.
- The heating element is fixed in a horizontal position. I think (and am currently testing the theory), that you can alter the cycling velocity of the fluid by changing the angle of the plane of the heaters. i.e. when they are aligned vertically, you get the fastest speeds, and near horizontal the slowest. That way you can alter the extension time, and thus PCR longer targets.
- LavaAmp seems to be vapourware, as http://lava-amp.com is down, and the blog hasn't been update since 2010
- Lastly, its not Open Source hardware and software. You can download my schematics and code right now, and build your own Polymerase Chain Reactor :)

Are you sure? [yes] / [no]

davedarko wrote a month ago null point

something went wrong with the formatting, I guess there is a break within the table.

And congrats to your working setup!

Are you sure? [yes] / [no]

David wrote a month ago null point

OK! all fixed now I hope.

Sometime today or tomorrow, I will upload the schematics and source code, so that other people can build the v0.2 prototype. That should be enough to verify that it works, and help identify any potential flaws or improvements in the design.

I have already moved on to using a single ATMEGA328P using V-USB to save the cost of a FTDI chip, but another option is to build a DNA Replication shield for the Arduino. The Arduino Uno already has the microcontroller, 3.3V converter for a reference voltage, and the USB interface I need. Any opinions? Would this be better than a more expensive standalone system?

Hopefully this is the kick the DIY BioHacking need to go mainstream. If I win the Hackaday prize, or make it to the top 3, I will next design a super cheap Gel Electrophoresis and Incubator system. That should be enough hardware to bring Recombinant DNA technology to the classroom and Hackerspace.

Are you sure? [yes] / [no]

Phil Howard wrote 2 months ago null point

Could you use a convection tube to control how the fluid flows? Maybe even a double helix heat exchanger, where the fluid flows up one helix, round a U and down the other.

Creating a more complex shape might permit a series of changing temperatures, largely by winding around warmer or colder water. A simple version would run back and forth between hot and cold sides in the right ratio to set the temperature.

Again it's all driven by convection so only the shape is necessary.

Are you sure? [yes] / [no]

David wrote 2 months ago null point

Yep, that's what I have in mind, but somewhat more straight forward.

Are you sure? [yes] / [no]

daveatfernie wrote 2 months ago null point

I'm also interested to see the viability of the project. I have built PCR instruments for the last ten years and usually the effort is put in to have homogeneity in the tube to ensure that the product has amplified properly. I could see the tube containing a lot of primer dimers. How are you intending to test the DNA once it has been amplified?

Are you sure? [yes] / [no]

David wrote 2 months ago null point

I'll PCR test fragments of various lengths and with various primer Tm's to test see if the principle is sound, and check by seeing if I get good gel bands. Then I'll clone some the fragments into vectors for sequencing, to make sure I'm getting real results. The idea isn't to have a general purpose lab PCR machine, it's to get a cheap machine for schools and hobbyists to try PCR, without having to spend hours slaving away in front of water baths.

Are you sure? [yes] / [no]

davedarko wrote 2 months ago null point

How long should the temperature transition times be? I don't think water will cool off that fast so you have to pull it out some how? Is it a crane which dips a cocktail of DNA and polymerase into hot water and different heights above the boiling pod? I'm just letting my mind spin here... interesting project anyways!

Are you sure? [yes] / [no]

David wrote a month ago null point

Sorry for not replying sooner! The times used in a standard PCR thermocycler are all most about the heating and cooling times. Standard 'fast' protocols use 20 seconds denaturing, 20 seconds annealing, and about 30 seconds for each thousand base pairs of target you want to copy. But, lab-on-chip devices that can heat or cool tiny specks of fluid seem to run nearly 10 times the speed. I guess a few seconds each for the first two steps, then 30 seconds per kbp is the best you can do?

Are you sure? [yes] / [no]

Insapio Limited Company wrote 2 months ago null point

Interested in seeing where you're going with this! Kary Mullis is my hero. Plus, hard to beat $1.

Are you sure? [yes] / [no]

David wrote 2 months ago null point

Sorry, but I have had to raise the price to comply with the competition rules. However, now there will be temperature and time tracking. It things get really complicated, it may even reach $10, but I'm trying my best!

Are you sure? [yes] / [no]