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

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!

V0.2 is ready to be replicated!  Build your own, and start replicating DNA.

The Eagle files have been linked to my Github account. It's a simple 1-sided board design, with no complicated or hard-to-find parts :)

Good news Everybody!

The next iteration of the Polymerase Chain Reactor v0.2 works!  Not only that, it works really well, with very good amplification of DNA in just 1 hour (thats actually faster than professional machines!)

First, here is the results of the Gel Electrophoresis of the experiment:

I used a plasmid containing the Green fluorescent protein gene as the template, and primers designed to bind to the beginning and end of the gene. On the right of the gel image is the DNA ladder, used to judge the sizes of the fragments. I have marked the the sized from 3000 base pairs in length and lower.

On the left is a very strong band of about 700 base pairs in length (the whole gene is 713 base pairs), and above that, there is a faint trace of the template plasmid, with a length of 4500 base pairs.

The size of the band indicates the amount of DNA it contains. The ladder DNA has calibrated amounts, and the 3000 base pair band contains 125 nanograms of DNA. As the PCR product band in 3-5 times as dark, the 5 microlitres of PCR mixtures I added contains about 400+ nano grams of DNA.

The total reaction volume was 180 microlitres, so we have a total production of about 15,000 nanograms, or 15 micrograms. Just how many copies is that? Well, plugging in our 714 base pair length and 15 microgram yield into this calculator, we get 34.575 pmol (pico moles). Multiplying this by the Avogadro constant give us a value of 20 trillion copies! Not bad for an hours work...

So, on to the details.

This time, instead of using a small reaction vessel, I used a loop of telfon tubing. It had an internal diameter of 0.5mm, and a wall thickness of 0.25 mm, and a length of 18cm. The tube's end were joined by pushing them into a short length of silicon tubing, after filling it with about 180 microlitres of PCR reaction mixture.

This time I used much longer 11W, 10 ohm ceramic resistors. I had hoped to use the groove in the resistors to hold the tubing in place, but the temperatures vary a lot! the outside of the resistor could be 95 Celsius, but the temperature inside the groove over 120C! So, I used some cheap aluminium grooved offcuts to act as heat distributors, and these gave a very stable 95C over the entire surface.

The second PID controlled heater was set to 72C. The aluminium rails were cable-tied to the heating resistor, and the TO-92 sized temperature sensor held in place by tucking it under the tensioned cable tie.

The telfon tube of PCR mixture was pressed into the two rails, and was held in place with some sticky tape. The reaction is driven by thermal convection, with the 95C heated side raising the liquid to the cooler, unheated tubing. The 72C side can't complete against the buoyancy generated by the 95C side, so the fluid falls through this section of tubing.

The unheated side is where the primer annealing takes place, and I measured its temperature at about 58C, a really great temperature for annealing most primers.

The reaction ran for an hour, giving the results described above! We have a working PCR machine!

Parts List

The cost to build your own is pretty good so far. I ordered my parts from Farnell, with the exception of the cheap Arduino Nano clone I had bought from China previously. For to build a single unit, you need to pay € 9.37, plus the cost of cable ties and some perf board. 

I have already done some cost calculations for orders of 2000+ units using surface mount parts, and including a custom PCB, I think the ~$5 target is doable. That will be the goal of this Hackaday Prize entry, but I think $50-$100 for a better machine with thermal fuses, a case and inbuilt user interface would be a great kickstarted project.

The first iteration of the PCR machine is finished. However, I've had negative results from the simplest formulation of the device.

The machine is comprised of 2x PID controlled heating elements, that generate a circular convection pattern in the 250 microlitre reaction tube. I used Herculase II polymerase in a 200 microlitre reaction, with primers designed to amplify out a 615 base-pair long gene. The target was the cAMP receptor protein, with primes of about 20 and 21 bp's and an annealing temperature of ~58C.

The v0.1 prototype uses perfboard and 10 ohm 4W resistors as a heating source. Although I used PID software, the low thermal mass of the heating element meant that Proportional control alone was enough for good temperature stability with no overshoot.

The lower heating element was set to 95C, and the upper to 50C for annealing. The 10 ohm The reaction was made for one hour, with samples taken every 15 minutes to measure the progression of the reaction. Sample were 5 microlitres, and were replaced with PCR mixture (without polymerase, as it digests primers at room temperature).

The 4 samples were loaded into an agarose gel for electrophoresis:

The ethidium bromide dye was used to visualise the DNA, and I used a 100 base-pair and 1000 base-pair ladder to judge the size of the PCR product.

The imaged gel shows the two measurement ladders, 100bp and 1k bp's on the left and right side respectively, but no product bands. This means that the PCR reaction did not happen. I have repeated the reaction a few times, with varying annealing temperatures, but haven't yet seen product bands with the v0.1 prototype.

The first parts have arrived, and PCR testing using this setup will commence this week. 

The initial prototype uses:

• An clone Arduino Nano
• 1x LM7805 voltage regulator
• 2x 2SK3561 N-channel mosfets
• 2x LM335 temperature sensors
• 2x 22 ohm 4W resistors
• various resistors and capacitors
• cable ties!

Of course using an Arduino nano will blow the $5 budget, even using very cheap clones bought online from China. So in parallel with the PCR experiments, I'm designing a new system based on the new ATTINY841 micro controller together with the V-USB serial library, as modified by Ray and described here: http://rayshobby.net/?p=7363.

I expect I will have to move to surface mount components to get under $5, but for the moment in using through-hole parts to get a working system up and running. I should mention that you'll have to bring your own power supply, with an output of 9v and about 2-3 amps, to get this thing running.

Got the video for the competition made; nothing like last minute pressure to defeat procrastination!

Hopefully all the parts for the first prototype should arrive in the next few days. Once I get  PCR working in the prototype, you can expect a progress report that includes electrophoresis gel pictures and many many more details.

I have done an initial test on single tube PCR, using a laboratory heating block set to 95C, with one side of the tube heated and the other cooled. On my first attempt, I tried heat in the side of the tube, but I didn't see any PCR product. On my second attempt,  with heated on the sloped bottom side of the tube, I saw a nice band after agarose gel electrophoresis. This was after a half hour of reaction time! 

I have now ordered a set of components for my first prototype. I will be looking at an Arduino-type setup, using either a ATMEGA328 or, if I can get the code optimised, I would like to use the ATTINY841, which should help keep the price low. I can't go much smaller, as I need at least 3 ADC's, 2 pins for serial (or V-USB), and 4+ DIO's.

I would like to use a JY-MCU bluetooth adapter for connecting to a server running on a PC, allowing a web interface to control the heating parameter of the PCR machine, but that would increase the price by a few dollars. Another option would be to use the V-USB interface, and have a wired connection, which would keep costs lower. Of course, I'm open to suggestions! Please let me know if you have a good idea on how to connect my PCR machine up *cheaply*. I need 2-way communication, to log temperature data and to set temperatures and temperature ramps. (yes, I'm looking at implementing Touchdown PCR :)

So, I guess people are wondering how this thing will work?

Well, this is how the cheap version will work. The much more complex version is far superior, and I will focus on that, once I get a working prototype of this simple version built.

Firstly, for the naysayers, convective PCR is still being researched in several science labs around the world, and the technique is proven, but almost always uses expensive Peltier effect devices.

To keep costs down, I plan on using boiling water, which at 1 ATM boils at 100C.  The heat will spread though a heatsink,  following a gradient from 100C to room temperature. By affixing a PCR reaction vessel to the point on the heatsink at 95C, we have our steady denaturation temperature. Induced convection in the vessel will cycle the reactants though the annealing and elongation temperature.

I have done some 2D modelling of a standard 0.2ml PCR tube, heated to 95C on one side, and heatsinked to room temperature on the other, with insulation around the other edges.

With side-heating and cooling, we end up with a large dead-zone in the velocity field in the bottom of the tube. This dead-zone also has a temperature below that needed for PCR, so this orientation is obviously not good.

With the tube oriented so that the sloping wall is in contact with the heatsink, we get much better circular convection throughout the entire tube. From the heat map, we see that the fluid reaches all the temperatures necessary for the PCR reaction. The centre of the tube shows a large region at around 70C, great for the elongation phase.

It's important to note that we not every molecule is going to follow the correct temperature sequence. But with exponential growth, only a subset have to follow bounce around the tube in the correct sequence  for the reaction to be successful.

So, with the initial modelling done, I will get a test system built, to see if the model work in practice. The next step is a prototype built. How will I keep the materials cost low? Well, firstly, I will use my thermal camera to find the position on the heatsink, so I don't need to build the temperature probe and serial connection just yet.

But the main costs for apparatus will be a few empty soda cans, some tap water and a tea light candle :)

Of course, the main issue with this technique is that the time and temperature cycles can't be easily controlled. However, there are ways around this problem, which I why I want to focus next on a more expensive, but much more useful design.

More to come soon.

So, I have done quite a bit more research into the design of my PCR machine. I can follow one of two options:

1) Make the cheapest possible PCR device. I estimate that this will cost under $1, but will be limited to replicating DNA fragments of up to about 200 base pairs. That might be enough for DNA fingerprinting, and really small genes, but its pretty limited. It will however be easy to make at home and in schools.

2) Make a useful PCR machine with a fixed extension time of about 30-60 seconds, enough for amplifying up to 2-3 thousand base pairs with a fast polymerase. This will need much more electronics, and probably end up costing around $30. It will be a project suitable for intermediate makers, but probably beyond that of most high school kids.

I'd love to hear some input at this stage, before I choose a direction to go with.