BME100 s2014:W Group5 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: Garrett Sciascia
Role: Mad Scientist
Name: Juan Bahena
Role: OpenPCR Technician
Name: Blake Marx
Role: WetWare Correspondent
Name: Danielle Hallaran
Role: Data Collection
Name: Naomi Walliman
Role: Team Manager



LAB 6 WRITE-UP

Computer-Aided Design

TinkerCAD

TinkerCAD is a web-based computer-aided drafting programme that allows the users to design three-dimensional objects. These objects can then be manipulated in size, shape and colour before being sent to a 3D printer and output as a real, physical object.



Our Design


NewLid2.png

We modified the design by manipulating the material used to build the OpenPCR machine rather than the shape or functionality of any given part.

One of the more frustrating parts of working with the OpenPCR was the difficulty we had in finding the optimal tight- or snug-ness between the heated lid and the samples underneath it. In changing the material used to build the side walls of the lid to something more transparent (yet still retaining the conductive properties of the metal), the user will not need to estimate so much when tightening the lid over the samples (or having it pop open after tightening the lid too far)




Feature 1: Disease SNP-Specific Primers

Background on the cancer-associated mutation

The single nucleotide polymorphism rs237025 was for a human with diabetes. A nucleotide is an organic molecule made up of a nucleotide base and a polymorphism is when 2 or more clearly different phenotypes exist in the same population of a species. This SNP is associated with the gene SUM04 TAB2 which stands or small ubiquitin-like modifier 4. This gene is located in the cytoplasm and modifies IKBA, leading to negative regulation of NF-kappa-B-dependent transcription of the IL12B gene. The non-disease allele contains GTG and the disease-associated allele was ATG. Therefore, a change in the G allele was the link to the disease.


Primer design

  • Disease SNP-specific Forward Primer: 5' T G A A C C A C G G G G A T T G T C A G
  • Reverse Primer: 5' C G T C T A G T C T A A G G C T A A A C

How the primers work: Primers are attached to the double-stranded DNA and tell the enzyme where to start copying in the PCR process. They are disease-specific primers due to their structure and their location of attachment.

Feature 2: Consumables Kit

One of the major issues we had with the consumables was the issue of SYBR green bleaching. Repeatedly covering and uncovering the entire set of samples got tedious after 20+ samples and merely putting foil over the top of the samples does not prevent reflected light from coming in through the bottom.

To fix this issue, we plan to re-package the consumables in the kit with a black, light-proof tube (or tubes) for the SYBR green dye. This will allow the user to leave the tubes uncovered without running the risk of ruining the test.

Another issue was the glass slides being used. There was difficulty with keeping the trials separate from each other as the solutions would splash around on the glass slide. It made it hard to keep track of what part of the glass slide had been used without discarding the whole slide and using a new one.

To fix this, the kit will come with labeled glass slides to keep better track of which part of the glass slide was being used. The glass slides will also have deeper divots to keep the solution from sliding around. This will decrease the likelihood of user error.

Feature 3: Hardware - PCR Machine & Fluorimeter

The contents of our system will contain the following:

  • Modified OpenPCR machine (see below)
  • Colored PCR tubes (see below)
  • Fluorimeter

The machine itself is more or less identical to the previous version of OpenPCR in terms of size, shape and function. The changes we made to the machine are intended to make it easier for the user to both place the samples correctly and to troubleshoot. This was accomplished by making parts of the machine transparent, and by modifying the side panel such that it was easier to remove and replace, respectively.

The new Open PCR machine incorporates a glass door to allow a clear, and safe window. The design improves the system by ensuring that the system is locked, visible from the user. This is an important detail, since the user does not want to leave the lid unsealed, and exposed to loss of heat. The new design starts with a glass material, polished with an aluminum interior. The purpose of the interior is to reflect and prevent heat from escaping. Next the interior is coated with a plastic, such as polyethylene, to add greater heat resistance. The idea of the design is based off the common microwave oven, which has a clear door, that is re-engineered for a higher specific heat capacity.

Slight cosmetic modifications will also be made to the tubes which hold the samples, SYBR green, buffer, etc. Given that the SYBR green bleaches when exposed to light, the tubes containing the dye are to be colored black so that the tubes do not need to be covered and uncovered repeatedly. Similarly, the rest of the tubes are to be color-coded in order to better visually represent what is in each tube. This will allow the user to determine the contents of each tube at a glance.

We had no issues with the fluorimeter so its design remains unchanged.

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

Specificity is defined as the probability that a person without the disease for a disease will test negative. Sensitivity is defined as the probability that a person with the disease for a disease will test positive.

The probability described in Calculation 3 was less than 1.0 and described the sensitivity of the system regarding the ability to detect the disease SNP. Calculation 4's probability was just under 1.0 and described the specificity of the system regarding the ability to predict the disease.

Based on the results, the OpenPCR machine better predicts that a person does not have a disease (true negative) rather than predicting a true positive result. Thus, the machine is more susceptible to false positives than false negatives. This characteristic could be an assuring result because the chance of a false positive can be eliminated by testing a sample multiple times (which is actually common practice in many DNA-testing facilities).