BME100 f2014:Group4 L6

BME 100 Fall 2014	Home People Lab Write-Up 1 | Lab Write-Up 2 | Lab Write-Up 3 Lab Write-Up 4 | Lab Write-Up 5 | Lab Write-Up 6 Course Logistics For Instructors Photos Wiki Editing Help
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: What Bayes Statistics Imply about This Diagnostic Approach The first and second calculations were quite close to 100% (1.00) which implies the reliability of the device. These describe the sensitivity and specificity of the machine to detect the disease and seeing as the high percentage, it implies a high rate of reliability. These first two calculations tend to see if the patient will be diagnosed positive or negative based on the PCR test. The high percentage for the first calculation test suggests that if the patient was tested positive, the patient has the disease. Similarly, in calculation two, if the PCR test was negative, the patient did not have the disease. Because this number was also near 1.00, there is fair amount of reliability. There are many sources of error that can be accounted in this experiment. Contamination could be one of those variables which could have caused an effect on the data. An example of this would be if when handling the DNA, the person handling could have accidentally contaminated the DNA causing inaccurate data. Another possible error can be attributed to the mislabeling of the PCR controls which would then affect the rest of the experiment as these were the comparative sources. There could also be a miscalculation during the ImageJ process while analyzing the fluorescence seeing the number of photos that needed to be analyzed in a short amount of time. Calculation 3 and 4 results were quite small in comparison to the 1.00 (100%) that the data was aiming towards, implying that the PCR was not reliable for predicting the development and diagnosis of the SNP associated disease. Calculation 3 deals gives the probability of a positive result in relation to the chance of having a positive diagnosis. Calculation 4 gives the probablity of the patient not getting the disease with a negative result. This implies that the diagnosis is not very accurate so there is a low chance of diagnosis before the onset of the disease. The same sources for error as calculation 1 and 2 can occur here in calculation 3 and 4. BME 100 Fall 2014 Home People Lab Write-Up 1 | Lab Write-Up 2 | Lab Write-Up 3 Lab Write-Up 4 | Lab Write-Up 5 | Lab Write-Up 6 Course Logistics For Instructors Photos Wiki Editing Help 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: What Bayes Statistics Imply about This Diagnostic Approach The first and second calculations were quite close to 100% (1.00) which implies the reliability of the device. These describe the sensitivity and specificity of the machine to detect the disease and seeing as the high percentage, it implies a high rate of reliability. These first two calculations tend to see if the patient will be diagnosed positive or negative based on the PCR test. The high percentage for the first calculation test suggests that if the patient was tested positive, the patient has the disease. Similarly, in calculation two, if the PCR test was negative, the patient did not have the disease. Because this number was also near 1.00, there is fair amount of reliability. There are many sources of error that can be accounted in this experiment. Contamination could be one of those variables which could have caused an effect on the data. An example of this would be if when handling the DNA, the person handling could have accidentally contaminated the DNA causing inaccurate data. Another possible error can be attributed to the mislabeling of the PCR controls which would then affect the rest of the experiment as these were the comparative sources. There could also be a miscalculation during the ImageJ process while analyzing the fluorescence seeing the number of photos that needed to be analyzed in a short amount of time. Calculation 3 and 4 results were quite small in comparison to the 1.00 (100%) that the data was aiming towards, implying that the PCR was not reliable for predicting the development and diagnosis of the SNP associated disease. Calculation 3 deals gives the probability of a positive result in relation to the chance of having a positive diagnosis. Calculation 4 gives the probablity of the patient not getting the disease with a negative result. This implies that the diagnosis is not very accurate so there is a low chance of diagnosis before the onset of the disease. The same sources for error as calculation 1 and 2 can occur here in calculation 3 and 4. 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 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 Faster Cooling Wires Materials for new PCR 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 PCR machine Fluorimeter Using 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. 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 PCR machine Fluorimeter Using 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.

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

PCR machine

Fluorimeter

Using 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.

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