Name: Fatima Sanchez Garcia
LAB 6 WRITE-UP
Overview of the Original Diagnosis System
Through the use of the polymerase chain reaction, it is possible to test the DNA of patients in order to determine if the disease associated SNP is in their DNA. The patient DNA was isolated and placed into a solution with a large amount of the base pairs, TAQ polymerase, and specific primers that correspond to the coding area of the disease associated SNP. All thirty-four lab groups were responsible for two patients, which means that 68 strands of DNA were tested. Many processes were implemented to ensure a minimal amount of error. In each lab group, each patient’s DNA was tested three times; this means that there were a total of 204 polymerase chain reactions run. In order to ensure that the PCR machine was working correctly, controls were included with the samples of DNA from the patients. These controls were known to be positive and negative, respectively, for the disease-associated SNP. Once these PCR was completed the results were analyzed with a fluorescent agent that was activated in the presence of the disease-associated SNP. The level of fluorescence was analyzed in an image analysis program called ImageJ, using images taken from an IPhone. For every trial three images were taken to ensure no error. This means that, with all of the lab groups taken into consideration, 612 images were analyzed in ImageJ. These are only images from the patients and don’t include the analysis of the controls and known concentrations. All of this data was then collected and compiled into a large spreadsheet for the entire class of BME 100 students. The data was fairly accurate most likely due to all of the efforts made to ensure that error was minimal.
What Bayes Statistics Imply about This Diagnostic Approach
Since the results for calculations 1 and 2 were close to being 100%, it implies that the reliability of the device is high. A person is more likely to determine whether they are positive or negative for obtaining the disease. Some sources of error may be that there was a miscalculation when analyzing the fluorescence in an image. In addition, the class results may have been miscalculated as well that led to a miscalculation in our Bayes' formula. Another source of error may have come from the machine itself.
The results for calculations 3 and 4 were very small which infers that the PCR is not very reliable for predicting the development of the disease. This implies that the patient will have a very low chance of diagnosis before the onset of the disease. The same errors as calculations 1 and 2 are the same for calculations 3 and 4.
TinkerCAD is an online product design service that allowed us to replicate the structure of the OpenPCR machine. Using the building blocks given to us through the website thingiverse.com, we re-created a PCR machine virtually. We then modified this basic structure to incorporate some of the changes we felt would better the device.
Our OpenPCR design made through TinkerCAD
The above design for our modified OpenPCR machine is very similar to the original. One of the changes we made to the overall structure of the device was placing the main heating block further into the machine. One of the problems we observed during the lab was the fact that the lid did not close completely due to the PCR tubes protruding from the block. By lowering this, we hope to eliminate this problem as well as speed up the heating process for the PCR machine. With the test tubes completely in the the heating block and heated lid they will heat up more quickly, another aspect we wanted to better.
Feature 1: Consumables Kit
Consumables Kit Includes:
- Box of Pipette Tips
- 8 labeled tubes containing 50 μL of primer mixture
- 8 labeled tubes containing 50 μL of DNA (positive, negative, and three samples of two sets of unidentified DNA)
- 6 labeled tubes of 80 μL calf thymus, all of differing concentrations
- Large PCR tube of SYBR Green (must be measured by individual)
- 2 Superhydrophobic Slides
For our new device, the consumable products will come premeasured to ensure accuracy. A set of eight PCR test tubes containing the predetermined 50 μL of the primer mixture (containing the TAQ polymerase) will be provided along with pre-labeled test tubes with 50 μL of DNA. Besides this, the kit will come with the necessary pipette, sterile pipette tips, and gloves.
The inclusion of premeasured consumables is meant to prevent any human error in the PCR process. With the tubes of 50 μL PCR mix, the individual will only have to pipette the DNA samples into the mix and not worry about the amount of primer mixture. Besides this, the test tubes, both for the primer mixture and DNA are labeled to prevent any confusion or cross contamination. The tubes for both the primer mixture and DNA will correspond.
Above is an example of the test tubes with the premeasured 50 μL of the primer. The test tubes will have corresponding labels to prevent any confusion. The example depicts the consumables for the positive sample.
Feature 2: Hardware - PCR Machine & Fluorimeter
- PCR Machine
- Lightbox with Built-in Camera
- Prepsoitioned Stand for Fluorimeter and Slide
- USB Cord
- Downloadable Image Processing Program (auto-generates the ImageJ results)
The consumables will still be processed through a PCR machine to denature the DNA. The fluorimeter will still be used to process the image of the DNA to look for any tell-tale signs of disease. Some adjustments made to the PCR machine include a quicker heating process for the PCR machine. The denaturing process is quite lengthy and the machine takes a while to go from the differing temperatures and on numerous cycles.
The fluorimeter, will come equipped with a built- in camera as well as the stand for the slide that is prepositioned to the correct distance from the camera. This will ensure that the image is taken form the correct location as well as remove potential error from the smartphone camera. Once the images are taken, a USB cord will connect the the fluorimeter to a computer and enter images. A downloadable program will then auto-generate the ImageJ processing, making the process more convenient and time efficient.
Lightbox with the slide portion of the fluorimeter 4 cm away from the built in camera