BME100 f2017:Group8 W0800 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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OUR COMPANY

Name: Janet Vargas
Name: Cassie Riggs
Name: Megan Pitts
Name: Chandler Ryder
Name: Jinpyo Seo

PCR People

LAB 6 WRITE-UP

Bayesian Statistics

Overview of the Original Diagnosis System

BME 100 split into 17 teams to diagnose 34 patients (2 patients per group). To find a certain DNA strand of the specific disease, a PCR machine along with necessary nucleotides and disease primers were used to undergo several DNA replication processes with repeated heating and cooling to amplify the disease-ridden strand. With this process, the non-disease DNA will be relatively small in amount. Then a fluorimeter and the software ImageJ were used to analyze the the solution samples. After analyzing the solutions, the data from all of the groups with the respective patients were gathered for analysis using Bayesian statistics. To prevent error in each steps, the PCR machine ran numerous cycles of heating and cooling, allowing for numerous DNA replications with the disease-ridden primer and the disease along with the nucleotides. In ImageJ calibration, all of the samples had pictures taken with the same setup with a couple of trials. In this BME 100 class, the 20 patients concluded positive, 48 patients concluded negative, and 2 patients came out to be inconclusive. There was a group that did not have any data for its respective patients, but the lack of data was not included. Challenges for the PCR lab in human errors such as not measuring the amount of solutions properly with the micropipettors and not keeping the set-up for the pictures correctly. Some calculation errors include not using the exact value. Although these errors may have popped up, due to the use of PCR machine running many runs, and with multiple trials, these errors were minimized.

What Bayes Statistics Imply about This Diagnostic Approach


Calculations one and two both regard the reliability of the PCR machine making an either positive or negative diagnosis; the frequency of a POS conclusion paired with a positive PCR reaction and the frequency of a NEG conclusion paired with a negative PCR reaction. The calculation one results, a POS conclusion paired with a positive PCR reaction, being nearly 80% frequency, implies that the reliability is considerably strong for an accurate positive conclusion. The calculation two results, a NEG conclusion paired with a negative PCR reaction, being about 97%, implies that the accuracy of a predicted NEG conclusion is even stronger than that of a POS conclusion.


Calculations three and four both regard the reliability for the system diagnosing the disease development; the frequency of a given "yes" diagnosis paired with a POS conclusion and the frequency of a given "no" diagnosis paired with a NEG conclusion. The calculation three results, a "yes" diagnosis paired with a POS conclusion, being just over 50% frequency, implies that base on the data the reliability is not quite strong. The calculation four results, a "no" diagnosis paired with a NEG conclusion, being roughly 85%, implies the reliability for a predicted negative is overall more accurate than a predicted positive.

One source of error that could have affected the outcome of the PCR process is that when using the flourimeter, the phone could have tilted or fell, and this would have led to inaccurate measurements. Another sources of error could have been during the flourimeter process a group switch the phone that they were using because this would have led to different results because each phone would have different settings and camera capabilities. Another source of error during the PCR process could have been when using the micropipetter, not all of the solution could have been released into the plastic vials. This would have led to less DNA being replicated and would have changed the final results for the reaction.

Intro to Computer-Aided Design

3D Modeling
The software utilized was Solidworks. In Solidworks, the height of the extruded cuts, in the heat plate of the PCR machine, was increased from 11 mm to 17 mm. The diameter of the cuts was then decreased from 5 mm to 3.75 mm, this was done to allow more slots to able to be fit into the heat plate. Then using the linear pattern icon within Solidworks, the extruded cuts were multiplied to go from 16 slots to 28 slots. To do this process though, the plan that we were using had to be reversed so that the newly replicated extruded cuts would align with the previous extruded cuts.

Our Design

SmallerversionG8.png
The PCR machine was edited to include 28 slots for the DNA to be placed into the machine instead of the previous 16 slots. Also the height of each of the extruded cuts was increased from 11 mm to 17 mm along with a decrease in each cuts diameter from 5 mm to 3.75 mm. This design would be helpful because it allows for more DNA to be replicated at once. Since it would be difficult to shorten the lengthy time required for the PCR process to take place, the PCR machine was edited so that more DNA could be made during each PCR reaction.


Feature 1: Consumables

The prepackaged kit will be easy for the user as it will have the most important items for the experiment. The kit will be made specifically for the PCR machine. The heating block for the PCR machine has been altered making the plastic tubes a different size. To make this easy for the user the kit will have the tubes that specifically fit the PCR machine. The DNA for the patient will already be in the plastic tubes to reduce waste. The other solutions (PCR mix, primer solution, SYBR Green Soltuion, buffer soultion) will be included in the package in their specific vial pre-labeled. The pre-labeling will make the experiment run easier for the user and help reduce human error for proper testing. The SYBR Green Solution will be wrapped in tin foil to eliminate the solution's exposure to the sun. The micropipettor will be included as well since this consumable is going to be used the most. The tips for the micropipettor will be placed in a smaller soft plastic box than the one used in class to reduce space within the kit. There will be enough tips for the experiment and a couple extra just in case. This is all that is needed for the PCR machine to run the machine to multiply the certain section of the DNA. There will also be the glass slides that will be used during the fluorimeter use, there will be 5 slides which can be used for 25 tests.

Example Photos:

Pre-labeled vials/plastic tubes

G8prelabeledtubes.png

http://www.biocompare.com/Nucleic-Acid-Electrophoresis/23398-Thermal-Cyclers-Thermocyclers-PCR-Machine/

Micropipette tips in soft plastic box for easy disposal

G8tipexample.png

https://www.universalmedicalinc.com/brand-non-sterile-ultra-micro-pipette-tip-0-5-20ul.html


Feature 2: Hardware - PCR Machine & Fluorimeter

In the new system, the PCR machine was updated while the fluorimeter remained the same. Compared to the old design of the PCR machine that could fit up to sixteen plastic vials, the updated design can now fit up to 28 plastic vials. With this change, the height of the PCR machine was increased, allowing for the depth of the vial slots to increase from 11 mm to 17 mm. Although this does not account for the 1.25 mm decrease in diameter of the vial slots, it is possible to fit more plastic vials within the machine, thus processing more samples within the same amount of time.

The design of the fluorimeter will remain the same, and within the system, will be used the same as paired with the PCR machine.