BME100 f2013:W900 Group3 L6

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BME 100 Fall 2013 Home
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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Name: Marissa Kulick
Name: Blake Woods
Name: Shaun Wootten
Name: Bryce Gonzales

Thermo Diagnostics


Computer-Aided Design


TinkerCAD was is a free online tool used to create simple 3D files that can be saved as .stl which can be used in more advanced programs or even 3d printed. On Nov 20th, we used it to quickly mock up an alteration to the concept of the flourimeter, which we thought was a little to big and bulky. Not only did the program allow us to include a visual document in the report, but it also allowed our team members to more effectively communicate the concepts that were difficult to explain in words.

Above is the redesigned PCR tubes that were both connected and reoriented using TinkerCAD software

Implications of Using TinkerCAD for Design

The redesign of the flourimeter was done using TinkerCAD software. The speed and simplicity at which a 3D technical image can be created allowed for team members to do more in less time. While the mock up wasn't anything that was ready to be printed and utilized as is, it did perfectly illustrate a concept and allow the team to all be on the same page in the redesign. If it was required, it would be possible to create a .stl file with enough precision to 3D print much of the flourimeter turning out a working prototype in less than a day.

Feature 1: Cancer SNP-Specific Primers

Background on the cancer-associated mutation

rs17879961 is a pathogenic mutation found on the 22nd chromosome on the CHEK 2 gene of homo sapiens. SNPs in the CHEK2 gene have been linked to inheritable cancer, as the gene normally produces a protein called checkpoint kinase 2, which functions in tumor suppression.

Primer design

  • Forward Primer: A C T C A C T T A A A C C A T A T T C T
  • Cancer-specific Reverse Primer: G G T C C T A A A A A C T C T T A C A C

How the primers work: Primers are designed sequences of DNA that allow for specificity in reproducing genetic material. The primers will bind around the gene of interest, before (5') desired section of DNA on both strands. Two strands are needed because while the two strands are compliments of each other, they are not identical. In addition, DNA has to be extended in the 3' direction by the primase, and therefore will need to sit over a separate portion of the DNA. By choosing a primer of approximately 20 base pairs, there is enough specificity to ensure that only the gene of interest is extended.

Feature 2: Consumables Kit

Included in the ThermoDiagnostics consumables kit will be all plastics; including the PCR tube holders, pipette tips, and the tube plate, also included is the micropipettor, and reagents. The micropipettor is used to easily ensure accurate measurements when transferring liquids from any tubes. Also added will be labels to our PCR tubes to avoid any confusions and/or mistakes as to which solution it is. To avoid human error in regards to the micropipettor, an instructional DVD will be included in the kit, and the instructions will reiterate the importance of watching this instructional video prior to any use of the micropippetor. This will eliminate any air bubble problems or difficulties obtaining all the liquid materials from the PCR tubes.

Because the SYBR green was sensitive to the light, making the results compose of discrepancies overtime we propose that we put the film used on sunglasses and coat it on the outside of the test tube. This would slow down the light that penetrates through the PCR tube and make the results more accurate overtime.

Feature 3: PCR Machine Hardware

The PCR Machine will be used to log the information recorded during the process. The PCR machine will be used in the experiment to duplicate the DNA to detect the cancerous segment of the DNA by replicating only the cancerous sequence composed in the DNA. The PCR machine will do this by denaturing the DNA at 95 degrees Celsius where the DNA is separated and annealed. After the annealing process begins primers that are predesigned to bind only to specific DNA, the cancerous DNA, attach onto the DNA segments. The DNA then goes through a synthesis where the DNA is heated and cooled and the DNA can stretch completely and then replicate.

The weakness found by the PCR machine is that the machine has to be hooked up to a computer to record and log the data, which could be possibly done easier if we could implement a Bluetooth device into the PCR machine and have the data logged to your smartphone wirelessly. This would also mean on-demand results with the constant stream of data going from the PCR and the smartphone making the system more efficient for the user with logging the data during the experiment.

Feature 4: Fluorimeter Hardware

The fluorimeter is used to emit a light and then, using the wavelength and intensity, measure the amount of a specific substance in a liquid. In this specific lab, the fluorimeter was used to detect if DNA had a specific cancer sequence. The decision was reached that the fluorimeter was not efficiently working to it's full potential. Instead, the fluorimeter was redesigned to have everything included in the hardware. The front wall of the fluorimeter box will lift vertically. On the inside of this wall will be the camera. When the box is closed, this camera will be level to where the LED light hits the desired drop that we want to take a picture of. This will eliminate the problem of light, the camera being a different distance from the drop with each picture, and the phone falling or being knocked over. This solution would simplify the problem, and result in clear, more efficient pictures being taken, which then leads to more sufficient data being recorded.

In this image, the LED light is illustrated by the blue pentagon, the droplet is illustrated as the green sphere on the slide, and the camera is shown as the gray square with the circle in the center. Also improved are the slides. They have been changed so that both sides of the slide are hydrophobic, to reduce the number of errors.

Bonus Opportunity: What Bayesian Stats Imply About The BME100 Diagnostic Approach

The output of calculation 3 posed to be a very small value and the positive predictive value for if the cancerous patient checks out with cancer or not diagnosed by the PCR-based diagnostic test. Because the answer to calculation 3 was very small this shows that the test was neither sensitive nor reliable. This is because the closer to 1 the value gets the more sensitive and reliable the test becomes in predicting if patients have cancer. The output for calculation 4 posed to be close to 1 and comprising of the test that would show if the patient does not have cancer, a negative predictive value. The test result of close to 1 means that the test is highly reliable in predicting cancer and is very specific and if the test value was very small the test would result as not reliable in predicting cancer and not very specific. Because of the test being highly reliable calculation 4 seems to be a good value to use when evaluating a patient.