BME100 s2014:W Group6 L6

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Owwnotebook icon.png BME 100 Spring 2014 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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Elizabeth Burkett
Rose Among the Thorns
Matthew Warren
The Handy Man
Samuel McCollum
Man in Charge
Frederick Sebastian
WetWare Master

                                                                                       Biomed Testing Corporation (BTC)


Computer-Aided Design


TinkerCAD is a user-friendly 3D design tool that allows users to make a 3 dimensional representation of their idea. Instead of using a pre-existing design, we used simple shapes that we could easily manipulate to alter the design of the PCR machine. We made these shapes to look like the basic designs we wanted to implement on our improved PCR machine.

Our Design

Pcr machine6.png


The new design allows for faster heating/cooling and therefore more accurate results. The box itself will be made out of wood-plated rubber as these materials are non heat conductive. There will also be a new ventilation system to make the cooling process of the PCR test faster. Additionally, there will be a total of three heating trays hence increasing efficiency. With the new modifications the total time required to conduct a PCR test will decrease.

Feature 1: Disease SNP-Specific Primers

Background on the cancer-associated mutation

Nucleotides are monomers (or the building blocks) of DNA, and when certain nucleotides have variations they are called polymorphism. This variation comes about due to the change a DNA makes. rs237025 is a type of polymorphism, more specifically it is a Single Nucleotide Polymorphism (SNP). It is found in homo sapiens (human beings) and specifically located on the 6th chromosome: 149721690. The genes that are associated with this polymorphism are SUM04 and TAR2 which are in turn associated with Type I and Type II diabetes, as well as coronory heart disease. The gene SUM04 is an acronym that stands for Small Ubiquitin-like Modifier 4. This gene works primarily in protein coding of small ubiquitin-like modifiers. An allele is a variation of a gene that finally determines the phenotype, and the three bases that have a disease associated allele contains the sequence ATG.

Primer design

  • Disease SNP-specific Forward Primer: 5' ATGATTGCTATGTGT 3'
  • Reverse Primer: 3' TACTAACGATACACA 5'

How the primers work: DNAs are made of a double helix structure and when they separate to replicate themselves, the primers will only bind themselves to the exact site on the DNA that has their complementary nucleotide bases. When the primers "realize" that the sequence they need to bind to is not perfect, they will not bind hence, the DNA amplification fails.

Feature 2: Consumables Kit

The BioMed Testing Corp. also supplies each purchase of a Thermal Cycling and PCR package with a consumables kit, containing the following regents:

SYBR GREEN 1 dye in disposable plastic tubes, variable tube size and quantity option provided.

• Standard PCR reaction mix containing DNA polymerase and dNTPs in disposable tubes.

• Calf thymus DNA, variable tube size, DNA concentrations and quantity of tubes option provided.

• Tubes for final reaction mix, sized to fit the patented BioMed tube trey.

• Tubes containing Buffer solution and demarcated by the letter B pasted to the top of the tube, variable tube sizes and quantities provided.

• Tubes with distilled water for dilution, variable tube size and quantity option provided.

• One standard sized container of disposable pipette tips

• One courtesy biohazard disposal kit (we are not liable for contagion outbreaks)

• A sharps container for disposing of glass slides.

  • Note: Micropipettes are not included.

Other consumables packages send light-sensitive reagents (the SYBR GREEN 1 dye) with a cheap and minimally effective reflective covering. BioMed Testing Corp. packages all light-sensitive reagents in tubing with a reflective coating over every surface, maximizing the accuracy of the fluorometry results. Additionally, the consumables kit list shows that BioMed supplies both a biohazard disposal kit and a sharps container. This is a part of our growing dedication to safety, minimizing potential harm to the consumer or to the environment. Of course, all tubes are color-coded based on their contents and identifiable based on a complementary and intuitive key relating each color to the corresponding reagent.

Feature 3: Hardware - PCR Machine & Fluorimeter

The new PCR and fluorimeter machine will have better circuit boards that will also assist with faster heating/cooling. The addition of a better venting system will also improve the time it takes to run the PCR test, which can be very unpredictable using the current system. The PCR machine will incorporate a fluorimeter in order to analyze amplified DNA samples, thus enabling the detection of disease. The fluorimeter is placed on the top of the tray, that has an attachment to support a cellphone to take pictures. This new attachment holds the cellphone in place thus not requiring adjustments from the user or careful placements of the phone. The attachment also has a mechanism that allows the user to allign their cellphones with the fluorimeter with ease.

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

This question may be restated as really asking for the distinction between sensitivity and specificity for a Bayesian test, and how these results correspond to the strength of the test. Sensitivity, in the context of this study, means the ability of the test to correctly identify a diseased patient as being diseased. This result was between half and 0.75, making it mediocre at identifying a correct positive. Specificity, in contrast, is the ability of the test to correctly identify a cancer-negative patient as being negative. This number was less than one but still quite strong. Essentially, this means that the PCR reaction and subsequent fluorometry are better at detecting the absence of the disease than the presence of the disease.