BME100 s2014:W Group11 L6

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BME 100 Spring 2014 Home
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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

Name: Christopher Stark
Name: Christopher Stark
Name: Kandace Donaldson
Name: Kandace Donaldson
Name: Jordan Nelson
Name: Jordan Nelson
Name: Cassiana Wright
Name: Cassiana Wright
Name: Yash Jain
Name: Yash Jain



Image:starkindustries.jpg


Image:ironpcr.jpg


LAB 6 WRITE-UP

Computer-Aided Design

TinkerCAD

TinkerCAD is a free online 3D design platform. It was used to create a rough image of the body of the PCR machine and add functional and cosmetic changes.


Our Design

Image:Open pcr pic1.PNG Image:Open pcr pic2.PNG

We chose a change to the design of the outer body of the Open PCR machine.

Our experience with the machine made us wish it was easier to open up the machine to examine its circuitry when things went wrong.

We redesigned the side panel to include a snap-in design with a small handle to allow the side panel to pop out easily but only when removed by hand.

Feature 1: Disease SNP-Specific Primers

Background on the cancer-associated mutation

SNP stands for "single nucleotide polymorphism". A nucleotide is the molecule that makes up the coding part of DNA. Its four types, A, T, C, and G, are arranged in a meaningful sequence that determines the proteins that will code to express each gene. A polymorphism is the occurrence of different forms of a gene that exist in high frequency in a population. Therefore, SNPs occur where there is a common variation of one nucleotide in a particular gene. The rs237025 SNP exists in humans, or Homo sapiens, and is located on chromosome six. It is associated with the SUMO4 and TAB2 genes and is clinically significant as a risk factor. This is because the SNP is associated with type 1 diabetes. SUMO4 stands for "small ubiquitin-like modifier 4". The function of this gene is to modify the protein IKBA so that it represses the expression of NF-kappa-B-dependent transcription of the IL12B gene, which codes for a cytokine that acts on natural killer cells. The allele (or version of a gene) that differs between SUMO4 genes consists of three nucleotides. The non-disease allele contains the sequence GTG. The disease-associated allele consists of ATG.


Primer design

  • Disease SNP-specific Forward Primer: 5' A A C C A C G G G G A T T G T C A A T G 3'
  • Reverse Primer: 5' A G T T T T C T A A T T G A G A A T G C 3'

How the primers work:

The forward primer is designed to complement twenty base pairs of the SNP containing the disease-associated allele. The reverse primer complements a sequence of the gene 200 base pairs away from the SNP. If both the forward and reverse primer are able to bind completely to those complementary sequences, the gene will be amplified during PCR. However, if one of the primers does not bind fully, PCR does not occur. Since the forward primer specifically contains the disease-associated allele in its sequence, it can only bind fully to DNA that also has the disease-associated allele. Thus, PCR will only occur if the DNA has the disease-associated allele, meaning this DNA will be amplified, and DNA with the non-disease allele will not.

References:
National Center for Biotechnology (NCBI) http://www.ncbi.nlm.nih.gov



Feature 2: Consumables Kit

1. Micropipettor with holder
2. Tube tray with telecoping lid
3. Pipette tips with box
4. Glass Slides
5. Reagents (PCR mix, buffer, SYBR Green I, Primers) in clearly labelled vials

The micropipette will be packaged in a box with a plastic mold to sit in along with a pipette holder for use between pipetting. The tube tray will be fitted with a plastic, telescoping lid so that during the experiment, light can be minimized from hitting the SYBR Green I as long as you are not directly under a light source. Additionally, the tray will have default labels such as A1, A2, A3, B1, B2.. etc. as well as a space to write your own label if preferred. Pipette tips will be packaged in plastic boxes, however, we will significantly reduce the amount of plastic used to make the box to make it cheaper.

The packaging plan addresses a few weaknesses. First, the telescoping lid will aid in protecting the SYBR Green I dye from light sources coming at an angle since this was a huge issue for many people. The reduced plastic pipette tips box also cut down costs. The micropipettor holder will help protect the pipette during periods of nonuse and make it even easier than it already is to use. And finally, the labels on the tube sheet should aid in keeping track of the tubes.

Feature 3: Hardware - PCR Machine & Fluorimeter

The PCR machine will largely be left as it was because it efficiently and inexpensively got the job done. The only addition will be a side "hatch" that will enable one to access the insides of the machine more easily for simple repairs. The hatch will be on the same side of the machine as was opened before, but instead of having to unscrew from the top and bottom and pry the side open, a snap-in design allows a simple pop-and-pull motion to remove the side. The design for a removeable tube tray that would be able to account for test tubes of different sizes in the top of the machine was also explored, but it proved to be inefficient and expensive.

This simple redesign addresses the major weakness of its difficulty to access the inside of the PCR machine. When we first were given a PCR machine there were loose wires, loose screws, and missing nuts. We had to open the machine several times and found it to be extremely tedious and potentially damaging to the machinery because we had to flip and rotate the device. We also noticed the machine had been dropped before, which was possibly for this very reason.

The fluorimeter redesign is also simple. The cradle will be fixed with screw-in bolts on both sides in order to fasten the phone into the cradle. This will allow the phone to be motionless in the cradle, which was a major weakness before.

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

Calculation 3 describes the probability that a person who has a positive final PCR test conclusion will develop the disease in question, type 1 diabetes. The results of this calculation were over 50%, but not by very much. Essentially, the PCR test was able to predict the presence of the disease more than half the time, but not much more. Calculation 4 describes the probability that a patient who has a negative final PCR test result will not develop the disease. The results of this calculation were close to 100%. The PCR test is very reliable in showing negative results for patients without the disease. Essentially, the test is highly specific, but not very sensitive. It is not highly reliable in actually predicting the presence of the disease.





Reference for Stark Industries Logo: Marvel Wikia http://marvel.wikia.com/Stark_Industries_(Earth-199999)



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