Kemal Ficici

Kemal Ficici

PCR Project-UPDATE — January 5, 2016

PCR Project-UPDATE

I haven’t been able to post very frequently on this blog, mostly because of the holidays and winter break, but I have still continued to work on the device. I completely finished the proof of concept device(I already had a prototype built , just a few minor tweaks here and there), and cleaned up my code a bit.

Here are a few photos:

IMG_4002.JPG

IMG_4001.JPG

As you can see, it’s currently a wire octopus nightmare, but the device works in general. Over the holidays, I haven’t been able to do much with the project.  I just changed the relay, as it was causing some issues, and cleaned up the code a bit. I’m planning on building an enclosure for the project, and I will definitely include a tube holder for the sample container. I’m going to order the supplies for the PCR(primers, polymerase, buffer), and gel electrophoresis, which I will use to verify that the reaction properly took place. I’ll also make sure to add an LCD display along with a few LED’s(makes things look cool), buttons and potentiometers. I’m going to improve the code and include a user interface where one can set the time for reactions and the required temperatures, along with some extra info that might be useful when operating the device. I might add a wifi shield to make it controllable via browser. I’ll also upload the code on github soon.

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New Project: Low-Cost PCR Thermocycler — December 18, 2015

New Project: Low-Cost PCR Thermocycler

I’ve been working on this project for a couple of weeks, and I’m still working on it. I want to design and build a low-cost PCR thermocycler.What it does is that it facilitates a PCR(Polymerase Chain Reaction). Using it we can take one(or more) strand(s) of DNA and make many identical copies. With these copies one can do many things. For example, we can identify a suspect in a crime scene, diagnose and treat certain illnesses, sequence a genome, and do many more things. We can also “swap” parts of DNA that were copied with a living organism’s DNA. But we are currently only concentrating on the replication process.

How does the reaction work? First, we get a DNA sample(Fairly easy to do as long as you have access to a microcentrifuge. If not, then you will need a lot of patience). To the DNA sample, we then add primers, polymerases, and nucleotides. We then get ready for the first stage of the reaction. We add the sample to a PCR thermocycler, this is where the magic happens. This reaction is completed in multiple steps.

    • First, we raise the temperature of the sample to around 98C for about a minute, causing the DNA strands to denature and basically unzip into single strands(think unzipping a zipper).
    • Then, we move on to the annealing stage. We then cool the sample down to 50-60C for about 20-40 seconds. This causes the primers(small fragments of DNA that attach to specific sequences of DNA) to bind to the DNA strands at specific sites and then the polymerases(proteins that complete DNA sequences by adding nucleotides) bind to the primers.
    • Now we get to the extension step. This is the part where the DNA starts getting copied. The sample is heated up to 78 degrees and the polymerases then start forcing nucleotides onto the DNA strands, eventually creating a whole DNA strand.
    • This whole process gets repeated many times, with each cycle doubling the amount of DNA.
    • If you want to read more on PCR, check the link here.

Video on PCR(crappy music included):

 

So what the device will do is just change the temperature of the sample(s) quickly and accurately.

So far, I have only made a basic prototype that just heats up and cools the sample.

PCR basic prototype
Fritzing Schematic of Circuit

[Note: I can’t upload the image of the device for some reason, so here’s a fritzing model. I will upload a new one as soon as I can.]

So, how does my circuit work? The whole device is controlled via an arduino. Analog data from the thermistor is read through pin A0, and converted to Fahrenheit(I should change it to Celsius) by using the Steinhart-hart equation.

Steinhart-hart
Source: Wikipedia:Steinhart-Hart equation

Then, based on the stage of the reaction its on, the arduino keeps the device at a stable temperature by switching the relay on and off. The relay is connected to a 24v cartridge heater on one side, and on the other side is a peltier element with a CPU cooler. The peltier device just pulls in heat from one side of the plate to another. I included the peltier device in the machine, since I want the heating/cooling to happen as fast as possible.

So far, the list of materials is:

  • 1 Arduino
  • 100K thermistor
  • 100K resistor
  • 5V DPDT relay
  • NPN transistor
  • 12V power supply
  • peltier device
  • CPU cooler(fan and heatsink)
  • 24V cartridge heater

The costs add up to around $70. If I fab my own board with the ATmega328P and also buy the components and parts from wholesale, I might be able to reduce the device to around $30. That’s pretty cheap, I guess.

 

So the basic device is finished. Once my other supplies(primer, polymerase, buffer, nucleotides) arrive, I’ll be able to test out the process. I’ll check to see if the reaction worked by running the sample through a gel electrophoresis experiment. Until then, I will upload the current code. I am also planning on making the circuit smaller, maybe even develop my own board. Once my 3d printer arrives, I will also make an enclosure for the device, as the whole thing is a horrific mess of wires. Next thing on my list is also to connect the device to the Internet, and develop a simple app to control it wirelessly(via smartphone/computer).