BME100 f2016:Group4 W1030AM L6
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Lab Write-Up 1 | Lab Write-Up 2 | Lab Write-Up 3
Lab Write-Up 4 | Lab Write-Up 5 | Lab Write-Up 6
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LAB 6 WRITE-UP
Overview of the Original Diagnosis System
In the BME100 10:30 class, 17 teams of 4-6 students diagnosed 26 patients total. In our small group of 4 people, we divided into 2 teams of 2. Each team divided the lab to increase efficiency. For example, one team completed the PCR Reaction and the other group conducted research on the human genome. Our group was given 2 patients' SNP. To prevent error, we used 3 replicates for each patient. Every group in the class used the following specific PCR controls: HEATED LID: 100°C INITIAL STEP: 95°C for 2 minutes NUMBER OF CYCLES: 25 Denature at 95°C for 30 seconds, Anneal at 57°C for 30 seconds, and Extend at 72°C for 30 seconds FINAL STEP: 72°C for 2 minutes FINAL HOLD: 4°C
We also used took 3 images of each specific PCR sample to compare to each other and get better data. On ImageJ we had to make sure we measured the RAWINTDEN came from the same amount of area from each picture. To get the best RAWITNDEN we also tried to keep the area in relatively the same area for each picture, avoiding bright spots.
One major challenge our group had was working with Image J. Originally we worked on 2 separate computers. This means that the area used to measure RAWINTDEN was different for the different images. During our calculations, we quickly found out that this badly affected the accuracy of our results. We then had to redo all the ImageJ measurements with the same area.
What Bayes Statistics Imply about This Diagnostic Approach
The results from the calculations (1&2) suggest that individual PCR replicates are usually quite accurate in determining the final PCR conclusion of a sample. When replicates tested positive for the disease SNP, the final conclusion also turned out to be positive almost 90% of the time. While not quite as accurate as one might expect, 9 out of 10 samples is fairly good.
The results from the calculations (3&4) suggest that the PCR test is very unreliable for detecting whether someone actually has the disease, and is somewhat unreliable for detecting if someone does not have the disease. When a positive final test conclusion occurred, the patient turned out to have the disease slightly less than 50% of the time. This means that the majority of the time, when the patient tested positive, they actually did NOT have the disease. This is extremely troubling for a diagnostic test.
Sources of Error
The second source of error could have been in our ImageJ measurements. As mentioned earlier, we originally used different areas to measure the RAWINTDEN. While we redid the measurements to fix the issue, the location of each area on the water drop varied. The different locations could have had more concentrations of light effecting our data for the Bayes Analysis.
The third source of error could have been in our calculations of the constants for the Bayes Analysis. The constants were supposed to be based off the conclusions from each groups' results. However, some groups did not turn in their data and had no conclusions. This gave us less data to consider into our constant which could have effected its accuracy. These somewhat inaccurate constants would negatively affect our Bayes analysis.
Intro to Computer-Aided Design
Feature 1: Consumables
In our design we have decided to keep all the original consumables. This includes the liquid reagents: PCR mix, primer solution, SYBR Green solution, and buffer. We will also include the plastic tubes, glass slides and pipette tips. However, our kit will present all these consumables in a user-friendly, easy to understand packaging. We will arrange each of the consumables in order of steps needed to complete the PCR. Preparing the PCR samples was a hassle. We had to put on the pipette tip,transfer PCR mix, discard the pipette tip, get a new pipette tip, and then transfer the DNA to the same tube. All these steps were on different trays and spread out around our table. With our design, all the steps are located in order on one tray. For example, the tray would have a row of tubes in this order: a pipette tip, PCR mix, pipette tip, DNA sample. Underneath this row would be the empty tube the mix is going into. This way the user simply goes down the row of tubes in order placing into the empty tube. We would also make a similar tray that does this for when we had to add SYBR green to the tubes. This makes it easier and more efficient to perform the PCR.
Feature 2: Hardware - PCR Machine & Fluorimeter
In our system of running PCR we have decided to keep the basis of our process of running the PCR the same in using the thermocycler the exact same. There is no outstanding weakness to the thermocycler and we see far too many strengths to the machine to make any significant changes to it. The machine that we decided to use in our process is the same style of thermocycler that we used in class It's lightweight which is a huge bonus in lab as many older PCR machines that you find in other labs can be twice to five times the size. In more devolved countries mobility may be a non-factor, bit in countries where labs and medical clinics are constantly on the move this would be a huge bonus to whoever may be running a PCR reaction to identify genetic and transgenetic diseases that may be found in a patient. Keeping it lightweight makes it easier to be mobile and simple to set up lab anywhere so that a scientist or whoever may be running the PCR can focus on the accuracy of their reaction and analysis of data to help the people they are running the PCR for. The thermocycler uses an Arduino microcontoller that is programmed on how the whole machine carries out the process which is a huge bonus. With a microcontroller such as the Arduino Uno being in our machine, the thermocycler can be reprogrammed in any way that a person running any PCR reaction can control the temperature changes that occur during a given PCR process. This is incredibly important in running PCR due to the fact that there are selective portions of DNA that denature at certain temperatures and when somebody is trying to isolate/amplify a very spesific portion of DNA the ability to change the max temperature of a cycle ran in the machine is critical to amplifying the portion of DNA being studied by whoever may be running the reaction. The use of a micro-controller also minimizes the amount of human error that could occur if the heating and cooing of the PCR were carried out by hand. The cooling system of the thermocycler is also fairly efficient even if the fan and the heat sink cooling system takes up a large part of the interior of the thermocycler, it is not necessary to change the design due to the ease of manipulation of the process due to the reprogram-ability of the arduino being such a large plus factor to the universal use of the thermocycler. The cooling system easily has the capability to cool items to the temperature of 160°C which at that point any human DNA being ran in PCR would definitely denatured enough to be subject to study.