BME100 s2015:Group1 9amL6
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LAB 6 WRITE-UP
Overview of the Original Diagnosis System
A total of 8 groups of 5-6 students each diagnosed 2 patients, thus 16 patients total per group. Each team tested for the designated SNP in the two patients. 3 different iterations were tested for each patient in order to decrease the possibility of error as much as possible. Error could result due to contamination of the samples, for example, from extra light that the samples were exposed to. Each sample was mixed with green fluorescent protein, or SYBR Green I solution, which indicated the amount of SNP positive DNA in the sample. The samples of the patients were compared to a control group, positive and negative. The control groups were also mixed with the SYBR Green I solution. After the samples were mixed with the solution, 80 μL drops of the final sample were pipetted onto slides. The slides were placed in a light box, and Images were taken of the drops in complete darkness so the fluorescence was captured. ImageJ software was used to analyze the images in order to determine the amount of SYBR Green I solution present. ImageJ determined calibration curves of the solutions with a known DNA concentration, which was used to determined the DNA concentration of the samples of the patients. A total of 3 images were taken per PCR sample as another way to reduce error. Ultimately, the values of concentrations helped determine which patient tested positive or negative for the disease. Class results showed a fairly equal amount of both positive and negative conclusions.
3 patients were ruled out in this experiment because of outlying/inaccurate data
The Bayes values for calculation 1 were very close to 1.00, and imply that the individual PCR replicates were reliable for deciding whether or not a patient had the disease SNP. Three possible sources of human or machine/device error may have been the deterioration of the solution when it was exposed to light during the light box portion of the experiment. Other errors may have been due to cross contamination, as well as the amplification process increasing the amount of contaminants also.
Using the TinkerCAD tool, we were able to fully recreate and improve the overall design of the PCR machine. This process was completed by creating the design of the original PCR machine first, and proceeding to add a heat block to the top of the machine and a number pad to the front of the machine. This was achieved simply by adding appropriately sized rectangular blocks to their appropriate locations.
1. One of the issues with the current PCR machine was its incapability to heat individual samples at different temperatures. There would be more potential with the device if it could have this ability. The heat block and cooling system could be upgraded to allow specific temperatures to be used for each sample slot.
2. In order to improve the portability as well as the functionality of the device, a interface was attached to the front of the machine. This would allow the user to choose whether or not he or she would like to use the computer interface or instead input temperatures and times using the keyboard on the machine.
The improved software in this new machine design is a rather simple addition and very easy to use. It is important to keep the changes fairly small because of the hardware interface changes, however the complexity of the machine overall will need to increase slightly because of the new added functionality of individual heating and cooling for each sample. In order to counter this, the user can choose the more complex function at the beginning of the PCR runs. This will allow the inexperienced users to have easy access to the device and also enjoy the increased functionality.
Feature 1: Consumables Kit
In the PCR kit, each individual consumables will be packaged appropriately. The liquid reagents will be packaged in separate plastic storage containers with rubber enclosing the rim of the containers as well as a cap to screw onto the containers. The pipet will be packaged in its own box with styrofoam inserts at the top and bottom to prevent the pipet from moving around in the box, preventing any possible damage to the pipets. The pipet tips will be packaged in a portable plastic box that makes it so the pipets are easily accessible and also prevention of any contamination.
In the consumables packaging plan, adding the rubber rim and cap prevents the possibility of any spills that could occur in transit. The addition of styrofoam in the separate box for the pipet prevents the pipet from moving freely in the box and results in its suspension in the box, preventing any damage if the box is dropped. The pipet tip storage container prevents contamination of the tips because they are prepackaged in a sterile environment, and ready for use when needed.
Feature 2: Hardware - PCR Machine & Fluorimeter
The PCR machine with the addition of a heat block and number pad will improve the overall process because the device will be portable and does not require any computer assistance, as well as accurate and individual heating of the samples
It is now possible for the Fluorimeter to be closed completely with fitted hole for a designated camera. This camera will fit appropriately in the hole, but in order to prevent any possible light that could still enter the box, an extra addition of a curtain over the camera will be added. The camera will be operated by a blue tooth remote that will allow the user to take pictures without disturbing the overall set-up and experiment. This camera can also be calibrated to determine whether or not a sample is negative or positive given a positive and negative sample. These changes would allow for the most accurate and reliable readings from the Fluorimeter.