BME100 f2013:W900 Group18 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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Contents

OUR COMPANY

Daniel McDermand
Daniel McDermand
Matt Hansen
Matt Hansen
Name: Nick Vale
Name: Nick Vale
Name: Dylan DeBruin
Name: Dylan DeBruin
Name: Kirstin Peters
Name: Kirstin Peters


The PertyPCR Cancer Diagnostics


LAB 6 WRITE-UP

Computer-Aided Design

TinkerCAD

In the lab on November 20th, the TinkerCAD tool was used to make changes to a previous design that was made using the same program. The file in TinkerCAD that we were required to redesign was an image of eight PCR test tubes. The TinkerCAD tool was used by our group to make changes that included adding a band on the tubes that served as a separation between hydrophobic and hydrophilic material, and color coding each one of the tubes in order to eliminate the need for labeling with a sharpie. In general, the TinkerCAD tool is used to create 3D designs, using a variety of shapes, scaling and orienting them appropriately until the desired design is made. The tool also allows changes to previous designs, which, in our case, is what we were able to do using the TinkerCAD program.

Image:PertYpatieZ.png


Implications of Using TinkerCAD for Design

The TinkerCAD tool is perfect for practical applications. The designs made in the program are easily transferrable to other programs that are able to print the design. In this case, since the 3D printers available to us use a plastic like material to print an object, the most practical application in this case would be printing one of the plastic components of the OpenPCR system. For example, camera holders could be mass produced using this program for two reasons: the TinkerCAD program is easy to use and it would be very easy to make something as simple as a holder, and the material that is used to print the objects would be sturdy enough to support the camera phone being used when taking pictures of the fluorimeter device. The TinkerCAD program makes it easy to design products in 3D, and the mass production of something as simple as a camera holder would be no problem for the program as well as the printer.



Feature 1: Cancer SNP-Specific Primers

Background on the cancer-associated mutation

Single nucleotide polymorphisms, also known as SNPs, are the most commonly found type of genetic variation in people. A nucleotide is the "building block" on the nucleic acid. It is phosphate group attached to a phosphoester bond, commonly known as an amino acid. In this lab, the SNP rs17879961 was examined. This SNP can found in the homo sapien species, and is clinically significant because it is a pathogenic allele. The rs17879961 SNP is located on the 22nd chromosome in 23 pairs of chromosomes that humans typically possess. The gene that is affected by the SNP is called CHEK2, which stands for checkpoint kinase 2. The danger with this is that CHEK2 serves as a tumor suppressor and regulates apoptosis, meaning that when the SNP alters the gene, it could lead to breast cancer, brain cancer, sarcomas and tumors.

Primer design

  • Forward Primer: 5'-TGTAAGGACAGGACAAATTT
  • Cancer-specific Reverse Primer: 5'-TGGGTCCTAAAAACTCTTAC

How the primers work: After the double-helix DNA has been unwound into two complimentary strands, the primers attach themselves to a target sequence and greatly contribute to the PCR process. During amplification, the target sequence is used as a template. Generally, a primer strand of about 20 nucleotides will only attach itself to one specific region on the DNA strand, only binding itself to that one part of the strand. On the other hand, primers who consist of less that 20 nucleotides often run into problems binding with more than one region on a DNA strand because there is less specificity in the sequence. If a primer is created to compliment a known sequence of the SNP, it will only bind to the segment of the SNP. This means that we can determine if an SNP is residing in a DNA strand through the use of PCR.



Feature 2: Consumables Kit

Overall, many parts of the consumes kit function well as they are and don't need modification. One major weakness that was observed, however, was that during micropipetting, drops of solution often gathered on the walls of the PCR tubes. To overcome this, our PCR consumables kit will have a set of additional PCR with a hydrophobic material on the upper majority of the tube. With that, all of the solution should gather at the bottom of the tube during pipetting, so that none of the solution is lost. This specialized set of PCR tubes could be used whenever very specific volumes of solution need to be mixed, and any error could significantly alter the results.

The consumables will be packaged in a normal manner. Specifically one that takes up the least space while still allowing each of them to be fully surrounded in Styrofoam for protection. The PCR tubes, micropipettor, and micropipettor tips will all be together in one box fully surrounded in Styrofoam for protection (primarily for the micropipettor). The primers and PCR mix will be packaged separately so that they can be refrigerated until the time of experimentation. All but the micropipettor are made of plastic, and therefore not significantly fragile, so no extensive protection is necessary beyond styrofoam in the box with the micropipettor.

Feature 3: PCR Machine Hardware

The PCR machine will have more holes for test tubes to get more data in during one sitting. Also, the new PCR machine will have an expanded LED screen to show more information on the test. The PCR tubes will be inside a consumable kit box and each tube will have a hydrophilic bottom and hydrophobic sides to help gather the solution at the bottom of the tube. This is so when the contents are poured out it will not stick to the sides of the tube and effect measurements and data. Also the PCR tubes will be color coded for easier identification because the old method of labeling with markers smudged and smeared off overtime making it easy to misread.


Feature 4: Fluorimeter Hardware

The main weakness with the Fluorimeter was having the phone stay properly positioned. Our improvement would be to make a new adjustable stand for the phone. This will lock the phone into place so it will not lose focus or move between photos. In addition and adjustable height so it can see any liquid or gel bubble at the proper level. Aldo adding adjustable positioning to tilt the phone to the perfect angle to get the best view and results.


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

[Instructions: This section is OPTIONAL, and will get bonus points if answered thoroughly and correctly. Here is a chance to flex some intellectual muscle. In your own words, discuss what the results for calculations 3 and 4 imply about the reliability of CHEK2 PCR for predicting cancer. Please do NOT type the actual numerical values here. Just refer to them as being "less than one" or "very small." The instructors will ask you to submit your actual calculations via e-mail. We are doing so for the sake of academic integrity and to curb any temptation to cheat.]

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