BME100 f2014:Group4 L6

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Perfect Consumer Robot: The PCR machine that everyone can use Name: Israel Ortiz Name: Dominique Stewart Name: Larrison Black Name: Zach Sledge Name: Shreya Ramkumar Name: Anyssa Iwamoto LAB 6 WRITE-UP Bayesian Statistics Overview of the Original Diagnosis System Through the polymerase chain reaction, DNA of patients can be tested in order to determine if the disease is connected to the SNP. In order to do this, a patients DNA must have been isolated and placed into a solution with base pairs, TAQ polymerase, and specific primers. After having many random students submit their DNA for a disease marker, thirty four different teams each went through the diagnostic process in order to analyze this DNA with each team responsible for two patients with a given three replicate DNA samples for a total of 68 strands tested. The data then went into a spreadsheet and when analyzed can determine whether the patient had a disease-associated SNP or not. In order to get accurate results and not misdiagnose, several precautions were taken. For one, each patient's DNA was tested three times for a total of 204 reaction trials in order to compare data and account for any human or computer error which could easily occur. In addition the work of dealing with so many patients was split up into several small groups with each group handling only two. As long as each group's work proved consistent, all the tests would be accurate and the testing would be finished with a comparatively low amount of work load and more in-depth care to precaution to each group's personal patients. Another thing that can often cause discrepancies is the PCR machine. The PCR machine must have had the same procedure and set-up for each trial and also include controls – positive and negative – to have a reliable source of comparison. Other mistakes can occur with the data analysis as interpretation is subjective. Therefore with pre-determined procedure and regulation, this can also prevented. The images taken from the experiment and were analyzed with a fluorescent agent activated with the disease-associated SNP measured through the only program known as ImageJ. Because three pictures were taken, if there was a bad image in that trial, the other two pictures could compensate to still produce accurate results. In this group, only two people worked together in order to analyze these pictures. Because the same person is analyzing these images, there is more accuracy between the different trials of each group. In order to preserve consistency between each group, specific instructions on how to analyze were provided with the activity. The data was then collected in a large spreadsheet and is fairly accurate due to the number of trials, consistency, and the many steps taken to ensure minimal error. Below is the class data: Calculations 1 and 2 were close to the wanted 1.00 (100%) therefore the data was accurate. The first calculation shows that if the patient is diagnosed positive then the diagnosis for the patient should be positive in that fact that they will have the disease. The second calculation deals with the same aspect but determines that if the PCR test was negative then the patient should be diagnosed as not having the disease. Because of the high percentage the data should be fairly accurate. Calculations 3 and 4 have a low percentage in comparison to the 1.00 (100%) that the test wants to reach. Calculation 3 deals with the probability that the patient will have a positive diagnosis if the tests come out positive and calculation 4 deals with the same aspect but with the negative fact that the probability of the patient not getting the disease in comparison with the negative test result. Because these values were so far from the 1.00, it implies that the patient will not have an accurate diagnosis in reference to when the onset of the symptoms appear. There can be many sources of error in this experiment. Some of this error can be human mistakes. For example mislabeling of the control groups could have mixed up the whole comparison data as the image proceeded to ImageJ. In addition on ImageJ, due to the number of people working on this program in so many different groups, each person could have drawn the analysis ovals differently which can cause the discrepancy between data. Finally another source of error can be attributed to contamination of the DNA. When the handler is handling the DNA, their own personal DNA could have contaminated the patient's DNA causing bad data. Computer-Aided Design TinkerCAD TinkerCad is an helpful 3D software which has pre-programmed shapes and tools which can be used to build a device much like staking legos on top of each other. In order to design the new system for this project, TinkerCad was used to model it. By importing the designs for the basic PCR machine, it can be used as a base template to make accommodations accordingly in order to develop a modified PCR system using the tools given on the sidebar. These tools on the sidebar helped with measuring parts of the system and aligning these parts together to create a virtual well-fitted machine. TinkerCad makes 3D modeling easy by providing easy step by step tutorials of the functions available. Our Design The PCR that we are designing incorporates many changes and, hopefully, improvements. The biggest improvement that we would like to focus on is lowering the time. Since the time that the DNA needs to be heated and cooled cannot be changed, we believe that by using a liquid cooling system, the time it takes to reach a certain temperature will save time. For example, the process itself is relatively short, but the current PCR takes hours to complete because the machine needs to reach the freezing and boiling points which are listed below as 100 degrees Celsius, 95 degrees Celsius, 57 degrees Celsius, 72 degrees Celsius, and 4 degrees Celsius. The liquid cooling system would need to be a separate part of the PCR so that the liquid doesn't affect the rest of the PCR parts if something were to happen so we added a small box that will be hanging outside the PCR. In addition to that, we want the PCR to be an independent machine so we are adding five buttons and a slightly larger LCD screen. The buttons that are being put onto the new PCR is a Start button that will turn the PCR on, then a Menu button in order to see select the functions that need to be changed, an up and down button to move around the menu and scroll through numbers for settings such as cycles, temperature, etc. The last button is an Enter button so that the functions can be plugged in. These buttons would operate as the computer program does. Instead of the USB port to connect to the computer, we want to change the PCR to be the common USB port for flash drives. The goal of this PCR is to have a faster reading and independent. Below is the research we have conducted thus far. We are going to delve into this research more, but this is what we have for now. Terminology Research PCR Cycle In a polymerase chain reaction, the machine conducts most of the work regarding the thermal cycle. Once the template DNA is put into the machine and the program settings are inputted, the first step of the thermal cycle is heating the template DNA to 95ºC for three minutes. The next step is called Denature or Denaturation, which is the process when the DNA separates into two complementary single strands from the one double stranded DNA. This phase lasts three minutes and then switches to the Anneal or Annealing phase, which has a lower temperature of 57ºC and also lasts thirty seconds. During this phase, the two primers bind to the complementary single strands. Next, the phase called Extend occurs at 72ºC and that lasts for thirty seconds where the Taq Polymerase generates new strands of DNA and attaches the primer to the base-pair. The next step continues to make the DNA larger with the replication of the DNA by the Taq Polymerase for three minutes. These steps are repeated 35 times, hence the name “thermal cycle.” Lastly, when the cycles are complete, the machine lowers the temperature to 4ºC to maintain the “product integrity.” This last step is called the Final Hold and stays that way till the tubes are removed. Heated Lid: 100°C Initial Step: 95°C for two minutes Number of Cycles: 35 Denature at 95°C for 30 seconds Anneal at 57°C for 30 seconds Extend at 72°C for 30 seconds Final Step: 72°C for two minutes Final Hold: 4°C Peltier The Peltier Effect occurs when heat travels on a current from one metal to a different metal where the heat sink helps keep the heat from returning to the other metal. A thermopile converts thermal energy into electrical energy, which is many series of thermocouples (two different conductors). Thermoelectric Cooling has a 10% efficiency due to the small size and lack of insulation. Required Parts: 1 Peltier 2 12V DC Fans 1 Heat Sink 2 Heat Sinks 1 12V Battery 1 Switch 1 Sheet Metal Net 1 Pair Alligator Clips 1 Meter Alligator Clips 1 Large Tube Thermistor A thermistor is a type of temperature sensor made of ceramic or polymer. The function of this resistor is to reach a temperature range from -90 degrees Celsius to 130 degrees Celsius with a higher precision. Liquid Cooling Systems Liquid-to-liquid: Requires a standby heat exchanger and downtime for the cooling system to clean, but it's the simplest and inexpensive. Closed-loop dry system: Utilizes an air-cooled fluid cooler in order to get the heat from pumped through rows of finned tubes and depends on the atmospheric dry bulb. Closed-loop dry system with trim cooling: Reduces reliance on city water as the coolant, which is expensive to purchase, so this is environmentally friendly as well as inexpensive to operate. During warmer times of the year, this requires a secondary coolant and piping. Open-loop evaporative system: This system uses a wet bulb and is inexpensive to use during warmer climates. This is not a good replacement for the PCR. Closed-loop evaporative system: This is a hybrid system that operates better than he wet bulb, requires less maintenance, but this deals with water so needs a drain, insulation, a heater, and pumps. Chilled water system: The chilled water system is like a refrigerator and operates the best over the wet bulb and the dry bulb, but is the most expensive. This also needs maintenance and requires more power to operate it. Enabling Button Functions In order to make the five buttons in our new design operate correctly, we need to know how to make the brains of the buttons. The buttons that are being put onto the new PCR is a Start button that will turn the PCR on, then a Menu button in order to see select the functions that need to be changed, an up and down button to move around the menu and scroll through numbers for settings such as cycles, temperature, etc. The last button is an Enter button so that the functions can be plugged in. In order for the buttons to work, an Arduino Board, a 10K ohm resistor, breadboard, hook-up wire and the buttons are needed. The reason for the buttons are to make the PCR function without the use of a computer or any help from other machinery. Faster Cooling Wires The wire that is mentioned numerous times is copper. Copper is "so cold that there is no resistance to electrical flow." Copper can have a temperature as low as -410 F. Feature 1: Consumables Kit The following items will be in the Consumables Kit – Parts List: Gloves SYBR Green with special black vials Taq Polymerase dNTPs Buffer Solution Micropipette 2 Refillable micropipette tip cartridges Primer Clear vials The new and improved consumables kit will come with clear plastic vials, a new and improved micro-pipette with two refillable cartridges. If requested more refillable cartridges can be bought with extra cost. In addition there will be an insulated container holding the reagents and another specific black insulated container holding the SYBR Green with five medium-sized black vials which can be separated from the packaging box. These re-attachable vials can help reduce contamination of the whole box as only a specific amount – enough to fill the vial – will be taken out at a time. In addition this will maximize the amount of trials that can be conducted as different teams can work on separate projects with the same SYBR Green base container. This way the black vials can be taken to a different location where the trial can be taken place and so another team would not need to locate where the SYBR Green is every time that group needs to re-fill as the SYBR Green will be in a base location. The black vials will also help keep out the light as the SYBR Green is being transported to different locations. One of the negative aspects about the kit that the group noticed during this experimentation was the number of pipette tips used in the lab as a whole. The pipette tip needed to be replaced as each different liquid is handled. This meant that the pipette tip needed to be inserted then the liquid dispersed then the tip dispensed leading to extensive amount of time placed into just the set-up of the pipette. Therefore, in this new consumables kit, a new type of pipette is introduced. This pipette would have a cartridge like system inserted into the pipette itself and a new button on top which when pressed would slide a tip to the front and secure it to the mouth of the pipette. Then the normal eject button on the pipette would be used to dispense the tip as normal. Feature 2: Hardware - PCR Machine & Fluorimeter The PCR machine worked well in the experimentation, but the PCR process involved a lot of pre-setup work. After inserting the test tubes in the machine and starting the cycles everything was automated but time consuming. A weakness we found is that a PC was required for programming the PCR machine. Another weakness was the long time it took to run the test tube cycles. To improve on those weaknesses we decided to create a PCR machine model that has a larger display screen and add buttons to do the programming on the actual PCR machine. The buttons would include a plus and minus button to increase or lower the value of any setting. Another button would be to switch a setting whether it is a temperature or number of cycle’s settings. Lastly an enter button to start the PCR process. Since the PCR process runs through a lot of cycles it is very time consuming which is due to the fact that the cooling system took way too long. Therefore the team came up with another idea to cool down the machine using a water cooling system which would work faster and would be connected to the side of the machine which would run water through the machine cooling it down much like how many video game systems cool down. This solution would shorten each cycle and decrease the total PCR process time significantly. In contrast, the fluorimeter was a complicated process with the many different variables that could affect the results. One of the weaknesses of gathering data from the fluorimeter, was configuring the distance between the phone deck and the place of the actual fluorimeter device. During the entire experimentation, when moving the black box to block out the light and the different trials with moving the glass plate around, the distance between the camera and the fluorimeter kept shifting. Team four therefore had to re-measure the distance between these two objects constantly wasting precious time trying to get consistent data. Another weakness that was noticed during the experimentation was the problem of fitting the phone into the phone stand to take the picture. Due to the latest phone updates as larger tablet-size phones are becoming the norm, the new phones fall out of the phone stands. Luckily team four’s group had a couple of older iPhones which could work in place of the latest iPhone6s which would fall down if placed onto the phone stand. In order to fix these problems, team four decided to attach an adjustable phone deck onto the fluorimeter. The phone holder can be adjusted to snugly fit different kinds of phones onto it by adjusting the width, height, and thickness of the holder which can be tightened to hold the phone in place. This phone holder will on the base will be connected to the base of the fluorimeter. In the space between a ruler measurement will be inscribed onto the machine. The phone holder will then be able to slide on the ruler base towards the fluorimeter or away and adjusted to the appropriate distance in order to take a picture.