BME100 s2014:T Group7 L6

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Name: Michael Penney
Name: Nolan Kern
Name: Nathan Flath
Name: Michael Tsarouhas
Name: Jordan Barajas

Lab 6 Write-Up

Computer-Aided Design

Using TinkerCAD

Our group contained a group memember who was familiar with TinkerCad so in order to learn about operating it we gathered around him. We learned how to manipulate, create and generate 3D objects through TinkerCad using the tools in the program. We then opened all of the open PCR pieces from the website and started assemblying them. We learned how to assemble the pieces into a whole by snapping them together. We then decided to improve upon the failure state performance and notification of the PCR machine.

Front view of PCR with new failure state alert
Close shot of new failure state alert

Why we use TinkerCad for Designing

TinkerCad is one of many designing softwares which allow for easy access and design of 3D objects. Instead of spending hours trying to properly convey the design of an object and its specifications by drawing one can send a couple of minutes using TinkerCad to generate a better image that follows the design specifications. TinkerCad also allows for easy modifications of their dimensions so make changes for production. TinkerCad is also 3D printer compatible meaning that you can streamline the production of the product straight from the design step and at the same time ignoring the need for companies or factories to make your product.

Feature 1: Cancer SNP-Specific Primers

Background information on the cancer-associated mutation

A nucelotide is an organic molecule that serve as the subunits of nucleic acids like DNA and RNA. Polymorphism in nature is when two or more distinct phenotypes exist in different DNA sequences. Single Nucleotide Polymorphism is when a single letter, i.e A, T, C and G, is different in a single nucleotide of an organism, in this case humans. The SNP rs17879961 causes the mutation that is related to cancer in humans and is extremely viral. The location of this SNP is on the 22nd chromosome in humans and directly affects a protien called CHEK2, also called checkpoint kinase 2. This protien regulates the production of cells in the body and when the mutation occurs the cells start dividing uncontrollably causing cancer.

Primer design

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

How the primers operate: The forward primer, from the patient, and the reverse primer, the positive, will bind to eachother if they contain the complementary SNP in the same location. Due to the nature of DNA assembly this will only occur if each one of the nucleotides has a complementary nucleotide on its partner sequence which allows for perfect accuracy when trying to identify the presence of rs17879961. The DNA were the sequence does not occur will not be double stranded and thus will not be replicated. This leaves the DNA that contains rs17879961 and our primer which will replicate thus producing enough of the phenotype to be detected.

Feature 2: Consumables Kit

In our kit will will include the following: pipette tips, SYBR green, glass slides, gloves and eye glasses. Each of these is essential for operating the PCR machine and thus is included in our delux starter package. The pipette tips will be placed in their standard box packaging, The SYBR green will be placed in PCR tubes that are secruely closed. The glass slide will also be placed in a standard box. Gloves will be placed in a small plastic bag and the same will happen occur to the eye glasses. All of this will be placed into our economical boxing discussed below.

Feature 3: Hardware- PCR Machine and Flourimeter

Our PCR machine will use the system discussed in feature 1 to determine whether the patient's DNA has the specific mutation in relationship to our positive control. Our machine will include a fail-safe light to denote if the machine is operating correctly. While testing our first device the decive had a critical failure: it would not start at all. Our aim is to remedy this problem by attaching a light to the PCR circuit which will denote if there is a problem with the machine. This will save time for the operator and help any repairs to be made faster and easier. Our PCR package will be the same as the standard PCR package with the only change being a light on the machine. The machine will be placed in a compact box for easy shipping and will include the four faces, all the screws, a pipette and the PCR software. All the pieces of the decive will be protected by styrofoam on the inside. An instruction kit will be included that follows FDA regulations and will include a password to a help site. The packaging will be wrapped in bubble wrap and placed in another box to added safety.

The Flourimeter will be included in a seperate box and is included in our package. This flourimeter works the same as a standard flourimeter, it will have a slot for a slide to be placed in and a light source to pass through any droplets. The flourimeter will also include a stand that is placed at a constant 9mm away to place a smartphone or camera on to take the picture, in a dark enviroment of course. The Flourimeter will be used to get the absorbance of the droplet and thus we can determine the concentration of our target sequence.