Difference between revisions of "Team:UMaryland/Design"

Revision as of 08:14, 18 September 2015

What is PCR?

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 polymerase through various temperatures. The reaction is started by heating the reaction mix to 95 degrees Celsius. The high heat overcomes base stacking interactions and hydrogen bonds which maintain the double helix, a process called denaturation. The machine then cools down to an annealing temperature in order for primers, short ssDNA oligos, to recognize selected DNA sequences, form duplex, and allow for polymerase to bind. Annealing is followed by extension, which is performed by the polymerase at its active temperature, typically around 72 degrees. The polymerase forms a daughter strand by adding nucleotides to the primer in the 5'-3' direction. I don't think this is necessary, especially not here. If you want to write how PCR works, put it in description. PCR is also a very complicated process so we'll need to invest a lot of space into it if you want to do it justice

Purpose

Although the process of PCR~~of amplifying genetic material is remarkable~~, the necessary hardware needed to do it is relatively simple. While different templates, primers, and polymerases can be used, a thermocycler capable of maintaining temperatures between 4 and 95 degrees is absolutely required.~~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~~ thermocyclers cost thousands of dollars, which is often prohibitively expensive for a DIY Bio effort. 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 valuable lab tool, enabling breakthrough research in more places around the globe~~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 consequence of expensive conventional thermocycling is the financial difficulty of bringing these machines into the classroom. PCR is an extremely important topic in biotechnology, but it is typically also one that requires one to "see it to believe it." However, schools often cannot afford to purchase thermocyclers due to their high cost. By manufacturing a cheap, DIY thermocycler that can be assembled at a low cost, we can help bring this technology into schools that would otherwise be unable to afford it.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.

Construction Hazard. Build at your own risk.

UMD DIY PCR

Our first design for a DIY PCR machine was modeled after a ~~more~~ conventional PCR machine ~~design~~. This first prototype used two pPeltier units, stacked on top of each other. These Peltier units would then heat a customized aluminum block that sat on top of the two units and held the PCR tubes. In order ~~for our system~~ to have feedback, we embedded a temperature sensor in the aluminum block to ~~detect~~measure the temperature of the wells ~~that held the PCR tubes~~. The sensor then reported back to an Arduino UNO, which ~~then~~ regulated the energy flow to the Peltier units, thereby regulating the temperature of ~~heating and cooling~~ the block ~~and tubes while closing the control loop~~. However, after much testing, this design proved to be ~~unoriginal,~~ expensive, and inefficient. While the conventionality of the design itself did not pose an issue, we realized that the parts used to assemble it were not as well-known or easily accessible to the general public, which we felt would take away from the possible applications of this machine. ~~In addition, although the price of this first prototype was relatively inexpensive in contrast to laboratory grade PCR machines, the price still ranged in the hundreds of dollars.~~Doesn't our machine now have an estimated cost of $200? ~~Lastly and most importantly~~, The greatest issue with our design was the inefficiency of the hardware; we found that the Peltier units were not able to quicklycycle through the desired temperatures ~~fast enough, e.g.,~~ causing the unit to~~would~~ take 5 to 10 minutes just to rise up to 95℃. After considering all of these factors, we began a redesign of our machine to better suit the needs of the ~~“Do-It-Yourself”~~DIY market.

The idea for our current thermocycler design first came into form when we found that our original prototype was not ramping up to the desired temperatures fast enough. ~~Because of this problem,~~ We thuslooked into other options for heating including a crude heat gun a.k.a. a hair dryer. ~~the machine and, in the process, 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℃ for PCR—in a matter of seconds. We then made a decision to suspend construction on the peltier-centered thermocycler in order to see how successful we could be ~~and how far we could go~~ with making a rapid PCR machine out of a hair dryer. Before this decision, we took into consideration the danger of working with a hair dryer, failure due to uncertainty that the machine could be effectively controlled, and, on top of that, having less time to work on it. ~~Nevertheless, we took the risk and are pleased to show you the results of our efforts.~~

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 <p>Our first design for a DIY PCR machine was modeled after a <strike>more </strike>conventional PCR machine <strike>design</strike>. This </strike>first </strike>prototype used two <strike>p</strike><b>P</b>eltier units, stacked on top of each other<strike,</strike><b>. These Peltier units would then </b>heat a customized aluminum block that sat on top of the two units and held the PCR tubes. In order <strike>for our system </strike>to have feedback, we embedded a temperature sensor in the aluminum block to<strike> detect</strike><b>measure</b> the temperature of the wells<strike> that held the PCR tubes</strike>. The sensor then reported back to an Arduino UNO, which<strike> then</strike> regulate<strike>d</strike> the energy flow to the <b>P</b>eltier units, thereby <b>regulating</b> the temperature of <strike>heating and cooling </strike>the block<strike> and tubes while closing the control loop</strike>. However, after much testing, this design proved to be <strike>unoriginal,</strike> expensive<strike>,</strike> and inefficient. While the conventionality of the design itself did not pose an issue, we realized that the parts used to assemble it were not as well-known or easily accessible to the general public, which we felt would take away from the possible applications of this machine. <strike>In addition, although the price of this first prototype was relatively inexpensive in contrast to laboratory grade PCR machines, the price still ranged in the hundreds of dollars.</strike><b>Doesn't our machine now have an estimated cost of $200?</b> <strike>Lastly and most importantly</strike>, <b>T</b>he greatest issue with our design was the inefficiency of the hardware; we found that the Peltier units were not able to <b>quickly</b>cycle through the desired temperatures<strike> fast enough, e.g., </strike> <b>causing </b>the unit <b>to</b><strike>would</strike> take 5 to 10 minutes just to rise up to 95℃. After considering all of these factors, we began a redesign of our machine to better suit the needs of the <strike>“Do-It-Yourself”</strike><b>DIY</b> market. </p>
 <br>
-<p>  The idea for our current thermocycler design first came into form when we found that our original prototype was not ramping up to the desired temperatures fast enough. <strike>Because of this problem,</strike> <b>W</b>e <b>thus</b>looked into other options for heating<b> including a crude heat gun a.k.a. a hair dryer.</b><strike> the machine and, in the process, disassembled a hair dryer to find out how the heating mechanism worked. To our pleasant surprise,</strike> <b>W</b>e found that the hair dryer was able to reach very high temperatures—much higher than the desired maximum of 95℃ for PCR—in a matter of seconds. We then made a decision to suspend construction on the peltier-centered thermocycler in order to see how successful we could be and how far we could go with making a rapid PCR machine out of a hair dryer. Before this decision, we took into consideration the danger of working with a hair dryer, failure due to uncertainty that the machine could be effectively controlled, and, on top of that, having less time to work on it. Nevertheless, we took the risk and are pleased to show you the results of our efforts.
+<p>  The idea for our current thermocycler design first came into form when we found that our original prototype was not ramping up to the desired temperatures fast enough. <strike>Because of this problem,</strike> <b>W</b>e <b>thus</b>looked into other options for heating<b> including a crude heat gun a.k.a. a hair dryer.</b><strike> the machine and, in the process, disassembled a hair dryer to find out how the heating mechanism worked. To our pleasant surprise,</strike> <b>W</b>e found that the hair dryer was able to reach very high temperatures—much higher than the desired maximum of 95℃ for PCR—in a matter of seconds. We then made a decision to suspend construction on the peltier-centered thermocycler in order to see how successful we could be<strike> and how far we could go </strike>with </strike>making a rapid PCR machine out of </strike>a hair dryer. Before this decision, we took into consideration the danger of working with a hair dryer, failure due to uncertainty that the machine could be effectively controlled, and, on top of that, having less time to work on it.<strike> Nevertheless, we took the risk and are pleased to show you the results of our efforts.</strike>
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 <h1><b>Design</b></h1>
-<p>Our design 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.
+<p><b>This goes in notebook, not design. What is your final design?</b><strike>Our design 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,</strike> <b>W</b>e began <b>by</b>working out how to wire the hairdryer so that we could regulate the heating unit and the fan separately.
 </br>
-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 in order to produce proper thermocycling.
+<strike>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 in order to produce proper thermocycling.
 </br>
-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 what these holes would do to the heat distribution(see picture below). To better distribute the heat we removed our tinfoil lid 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.We are also in the process of milling aluminum with certain specifications in order to better regulate heat transfer.
+At this point, we tried to perform our first PCR reaction<b>. U</b>nfortunately we soon found that we had melted our tube. We learned that the machine had difficulty with evenly distributing the heat<strike>, since the tin foil was a rudimentary cover with holes punched into it without a proper understanding of what these holes would do to the heat distribution(see picture below)</strike>. To better distribute the heat<b>,</b> we removed our tinfoil lid and replaced <b>it</b> with with a <b>cut</b> 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. <strike>For a better understanding we are currently in the process of modeling the heat transfer within the can to better design the apparatus.We are also in the process of milling aluminum with certain specifications in order to better regulate heat transfer.  </strike>
 </br>
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 </br>
-  <p>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 our machine worked.
+  <p>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 <b>moving</b><strike>convected </strike>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 our machine worked <b>ONCE</b>.
 </br>
 <h1><b>Hardware</b></h1>
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 <h1><b>Problems and Current issues </b></h1>
-<p> We have had one successful amplification with our machine however we understand that repeatability is a vital component of all lab work and currently we are attempting to make our device repeatable. From our early days of testing we found that peltier units were not powerful enough to enable PCR tube to reach 95 degrees. Although conventional PCR machines use these units frequently they are often specialized and tailored made to perform PCR. With this tailoring comes a high price tag that does not suit the DIY market, and so we found a solution in the form of a hairdryer. The fan and heating element of a hairdryer provide a control scheme that enables for cycling of temperature rapidly and accurately and they are relatively inexpensive. We have found that developing a housing for the PCR tubes and enabling even heat distribution is challenging. We often have found that our temperature sensor and the pcr reaction tube are not at the same temperature and degree of difference is a delta of over 10 degrees celsius. We are currently working of milling a block of aluminum with better and more consistent heat transfer properties, and modeling the heat transfer within the can. Our ambition is that this will enable better control of temperature within the device.
+<p> We have had one successful amplification with our machine however we understand that repeatability is a vital component of all lab work and currently we are attempting to make our device repeatable. From our early days of testing we found that peltier units were not powerful enough to enable PCR tube to reach 95 degrees. <strike>Although conventional PCR machines use these units frequently they are often specialized and tailored made to perform PCR. With this tailoring comes a high price tag that does not suit the DIY market, and so we found a solution in the form of a hairdryer. </strike><b>On the other hand, t</b>he fan and heating element of a <b>cheap </b>hairdryer provide a control scheme that enables for <b>rapid</b> cycling of temperature<strike> rapidly and accurately and they are relatively inexpensive</strike>. We have found that developing a housing for the PCR tubes and enabling even heat distribution is challenging. We often have found that our temperature sensor and the pcr reaction tube are not at the same temperature and degree of difference is a delta of over 10 degrees celsius<b>It is therefore NOT accurate, as described in previous sentences</b>. We are currently working of milling a block of aluminum with better and more consistent heat transfer properties, and modeling the heat transfer within the can. Our ambition is that this will enable better control of temperature within the device.
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