Polymerase Chain Reaction or PCR is a common tool used in the field of biology to amplify DNA or RNA. Invented by Dr. Kary Mullis, PCR is conducted trough cycling DNA, primers and enzyme through various temperatures. Generally starting with a value near and above 90 degrees Celsius; used to break the Hydrogen bonds between double strands a process called denaturation. The machine then cools down to annealing temperature, with values near 50-60 degrees, at this point primers are able to attach to the template strand of DNA. This stage is then followed by extension temperature, around 72 degrees, at this point the polymerase is able to extend and add nucleotides to the primer.
+
Polymerase Chain Reaction or PCR is a common tool used in the field of biology to amplify DNA or RNA. Invented by Dr. Kary Mullis, PCR is conducted trough cycling DNA, primers and enzyme through various temperatures. Generally starting with a value near 95 degrees Celsius; used to break the Hydrogen bonds between double strands a process called denaturation. The machine then cools down to annealing temperature, with values near 50-60 degrees, at this point primers are able to attach to the template strand of DNA. This stage is then followed by extension temperature, around 72 degrees, at this point the polymerase is able to extend and add nucleotides to the primer.
Polymerase Chain Reaction or PCR is a common tool used in the field of biology to amplify DNA or RNA. Invented by Dr. Kary Mullis, PCR is conducted trough cycling DNA, primers and enzyme through various temperatures. Generally starting with a value near 95 degrees Celsius; used to break the Hydrogen bonds between double strands a process called denaturation. The machine then cools down to annealing temperature, with values near 50-60 degrees, at this point primers are able to attach to the template strand of DNA. This stage is then followed by extension temperature, around 72 degrees, at this point the polymerase is able to extend and add nucleotides to the primer.
Although the process of amplifying genetic material is remarkable, the hardware needed to do it is relatively simple-- all that is required are three different temperatures which are maintained by the machine, enabling the enzymes and template to do the work of PCR. Current PCR machines cost thousands of dollars, and although there exists open source, DIY PCR machines, their costs still range in the hundreds of dollars. Here at the University of Maryland, we thought that that was an absurd notion. PCR, because of its simplicity and utility, is a robust tool for the diagnosis of many diseases both in the developed and developing world. Making the device cheaper would give more people accessibility to this platform. Accessibility enables further innovation and development of novel methods for disease detection and this in turn enables better and faster diagnosis and treatment both in the developed and developing world.
Another major advantage of "cheap" is education. Here at the University of Maryland, we acknowledge that iGEM is a competition, however we also understand that this competition is also a collaboration. It is an opportunity for all of us to learn from one another and serves as the foundation for future discovery, innovation, and new projects. We hope that our work with the PCR machine will inspire many more teams to tackle designing hardware. We hope that our current collaborations with Duke University foster better and more innovative projects from both of our teams. And most important, we hope that our efforts will be able to inspire the future generation of iGEMer's and the newest members of the iGEM community; high school students.
I remember, along with my fellow teammates, learning about PCR by cutting up little paper nucleotides and putting them into a brown bag and then having our hands act as the "polymerase" that would pluck the nucleotides out and match them with the template strand we were given. I remember taking away very little from this "lab" other than a few paper cuts. In subsequent years, I went through a few internship programs where I was able to learn in greater detail the steps of PCR, eventually learning how to design primers, program the machine, and setup my own reactions. However, I believe that if we truly want to bring synthetic biology to the public, we have to allow them the opportunity to actually do PCR, not through a paper bag which is conceptual understanding, but a real reaction where the end products are the real deal, actual amplified DNA. We still have a ways to go... the enzymes have to become cheaper pipettes need to become cheaper, but designing a below 50 dollar PCR machine is the first step in this endeavor.
Our first design for CHIP and employed, in many respects, a more conventional PCR design. CHIP utilized two peltier units below an aluminium heating block to heat the PCR tubes sitting inside the block. We used a temperature sensor to detect the temperature of the wells in which the PCR tubes were housed. The sensor then reported back to the Arduino unit, which regulated the energy flow to the peltier units, thereby heating and cooling the block and the tubes, and close the control loop. However our first design for CHIP proved to be unoriginal, expensive and inefficient. The design was conventional which in itself did not pose an issue, however, since these parts were generally not easily accessible to the general public we saw a problem going forward with this design. In addition although the price of the first PCR prototype was relatively inexpensive in contrast to laboratory grade PCR machines the price still ranged in the hundreds of dollars. The largest issue with our design was the inefficiency in the hardware; we found that the peltier units were not able to cycle fast enough. The unit would take a couple minutes to rise to 95 degrees. After considering all of these issues we began a redesign of CHIP to better suit the needs of the do it yourself market.
Our second thermocycler design, was mostly made out of a salvaged hair dryer. We came about this idea when we found that CHIP was not ramping up to the desired temperatures fast enough. Because of this problem, we looked to other options for heating the machine and disassembled a hair dryer to find out how the heating mechanism worked. To our pleasant surprise, we found that the hair dryer was able to reach very high temperatures—much higher than the desired maximum of 95 degrees Celsius for PCR—in a few seconds. We then made a decision to pause construction of CHIP in order to see how successful we could be at making a rapid PCR machine out of a hair dryer. We knew that working on the hair dryer would be much more dangerous and was risk since at the time we were unsure if the machine could be controlled to effectively cycle and amplify DNA.
The design of CHIP started when we bought a hairdryer in the hopes of using the heating unit as part of our first PCR machine. However, as we were dismantling and testing the hairdryer, it became apparent to us that the heating system inside the hairdryer could reach the necessary temperatures independent of the peltier units already in use. With this in mind, we began working out how to wire the hairdryer so that we could regulate the heating unit and the fan separately.
After a lot of soldering and reworking the internal safety measures inside the hairdryer, we were able to wire the system so that we could turn the heat on and off while running the fan continuously. Using autoclave tape, we secured a sheet of aluminium foil to the top of the heating unit of the hairdryer. The outer casing of the hairdryer had been removed. We placed a heat sensor inside the tin to measure the temperature of the air inside the machine. By wiring the heat sensor to the arduino we were able to receive input/feedback from the sensor and adjust heating of the device to maintain our desired setpoints. We were able to regulate the heat of the machine and CHIP now thermocycled.
At this point, we tried to perform our first PCR reaction, unfortunately we soon found that we had melted our tube. We learned that the machine had difficulty with evenly distributing the heat, since the tin foil was a rudimentary cover with holes punched into it without a proper understanding of when these holes would do to the heat distribution(see picture below). To better distribute the heat we removed our tinfoil led and replaced with with a soda can. This can was designed with evenly spaced holes enabling for better heat distribution. Although we did not and still have not modeled the heat transfer of between the can's surface and the convection heating generated by the hair dryer, we were able to experimentally conclude that the heat distribution was more even across the can than the tin foil. For a better understanding we are currently in the process of modeling the heat transfer within the can to better design the apparatus.
After construction of the can based cover we tried PCR once more and still found that the reaction did not occur. We assumed that the heat sensor might have been an issue,; the sensor was exposed to the convected air and was relaying information about the air temperature instead of the temperature inside of the PCR tubes. This meant that our feedback system was not accurately responding and controlling the temperature inside of the PCR tubes. Assuming the temperatures inside the machine were not representative of the temperatures inside the PCR tubes, we put the heat sensor inside a PCR tube with mineral oil and placed this inside one of the holes. We ran another PCR reaction, ran the products on a gel and saw a large band of the correct size, indicating that CHIP worked.
