BME100 s2014:W Group14 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



Our Team:

Name: Jose Duran
Name: Jose Duran
Name: Shawn Garcia
Name: Shawn Garcia
Name: Christian Keefer
Name: Christian Keefer
Name: Karthik Puncha
Name: Karthik Puncha
Name: Austin Tielke
Name: Austin Tielke

Our Product: The Thermal Cycling Spectrum

  • A new medical system that incorporates a modified OpenPCR machine and a modified fluorimeter


LAB 6 WRITE-UP

Computer-Aided Design

TinkerCAD

TinkerCAD is a tool that is used to create 3-D digital designs of objects and other 3-D printing compatible items. In class, each member was required to register (for free) on TinkerCAD. Two members of Wednesday's Group 14 were required to take at least 7 lessons online in order to learn how to maneuver throughout the program and the various tools. For the class period, our lab group was designated to select a specific portion of the OpenPCR machine and improve it in a way that resulted in a positive manner.

Our Design: OpenPCR Machine Modifications



PCR & Co. has performed several modifications to the original OpenPCR machine in order to propel its medical product system to the top of the competitive medical device market. The new Thermal Cycling Spectrum system will include an improved rendition of the original OpenPCR machine (as well as an improved rendition of the fluorimeter, which will later be discussed) that performs at a level that no other machines can compete with. The Thermal Cycling Spectrum system will feature a new OpenPCR machine that offers a wide spectrum of capabilities, allowing users to perform experiments smoothly without any altercations nor difficulties.

In short summary, this new PCR machine design features several adjustments that further enhance the user interface experience; thus, providing an ultimate experience that allows experiments and lab investigations to run smoothly, as well as more independently. The new OpenPCR machine aspect of the Thermal Cycling Spectrum system features a modified lid fastening system, an internal system accompanied with an external port system, and a modified docking station for a variety of PCR testing tubes. Further information will be provided for these modifications in the following paragraphs.

Feature 1: Disease SNP-Specific Primers

Background on the cancer-associated mutation

Nucleotides are organic molecules that serve as the monomers of nucleic acid, and are also commonly known as the building blocks of nucleic acids. Polymorphisms are coined terms for when two or more clearly different phenotypes exist in the same population of a species. For example, a DNA sequence - A, T, C, or G - in the same genome differs between members of a biological species or paired chromosomes. Short genetic variations are made up of single nucleotide polymorphisms (SNP's). After researching the SNP rs237025, it has been found in single nucleotide variations of the Homo sapiens (human) species. It is located on chromosome 6, and has no clinical significance; however, it has been linked to the health-related illness diabetes. The gene SUM04 stands for small ubiquitin-like modifier 4, which are attached to proteins and control the target proteins' sub-cellular localization, stability, and activity. Alleles are one of a number of forms of the same gene; they produce different effects. The disease associated allele for rs237025 is A-T-G, different from the non-disease allele containing G-T-G.

Primer design

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


Primers
A primer is a strand of nucleic acid that serves as a starting point for DNA synthesis, which is commonly experienced during the polymerase chain reaction. The enzymes that catalyze this process can only add a few nucleotides to an existing strand of DNA; however, it can only occur if there is a disease-specific primer. Forward primers will completely bind to disease-associated templates while reverse primers will perform the same function to their complimentary templates. When primers cannot completely bind to their respective templates, a polymerase chain reaction (PCR) cannot occur. If there are no disease-associated alleles, primers will not be able to function properly because there will be no disease-associated allele front- or back-end to bind to.


Feature 2: Consumables Kit


PCR & Co. has also modified the consumables kit that arrives with each Thermal Cycling Spectrum machine. Similar to the original consumables kit, this kit will be assembled prior to experimentation. The consumables kit will include the following:

  • PCR tubes
  • PCR reaction mix solutions, 50 μL/tube; contains Taq DNA polymerase, MgCl2, and dNTP's
  • DNA/Primer mix solutions, 50 μL/tube, each contains a different template DNA, and same forward & reverse primer
  • Micro-pipette tips, each only used once (dispose afterwards)
  • SYBR Green I Dye, quantity selected upon purchase
  • Biohazard bag: to dispose waste into
  • Kits may also include a 200 μL micropipettor, if the special edition consumable kit is purchased


These products will continue to be plastic in order to promote its consumable aspect. Each portion of the consumables kit will be pre-packed in its own respective compartment with respective color coding, along with a color coding key that provides the correct identification of each primer and PCR mix solution; thus, no products are mixed or lost.

The SYBR Green I dye will be placed in specific light-prevention PCR tubes in order to prevent any fraction of light from bleaching and harming the effectiveness of the dye. This method is similar to how beverage companies use darker (brown/black) glass bottles rather than lighter (green) glass bottles in order to prevent light from affecting the material within the bottle.

The PCR tubes that require specific identification (for the Thermal Cycling Spectrum machine) will be accompanied with small, blank circular stickers. These stickers allow for easier user interface while writing ID's or abbreviations, and will be placed along the side of each respective PCR tube; if the stickers are placed on the top of the lid of the PCR tubes, each sticker's adhesion may be removed due to the heating lid of the Thermal Cycling Spectrum machine.

Feature 3: Hardware - PCR Machine & Fluorimeter

The Thermal Cycling Spectrum System
The Thermal Cycling Spectrum complete system will include one (1) consumables kit, one (1) modified and newly updated OpenPCR machine, and one (1) modified and newly updated fluorimeter. Despite the consumables kit, assembly will be required for the OpenPCR machine and fluorimeter if the consumer does not select the pre-assemble option. If selected, this option will be performed in the PCR & Co.'s warehouse; however, an additional fee will be applied to the overall purchase. With all three major components included with packaging, consumers will receive the entire Thermal Cycling Spectrum system.

PCR Machine Modifications
This device features a new PCR tube holder that offers different size holes in order to allow different sized PCR tubes to be inserted smoothly. This allows several types of reactions to occur simultaneously during the several-hour thermal cycling period. Next, the new Thermal Cycling Spectrum features a new integrated internal system that blows the original OpenPCR machine out of the water. This internal system does not require additional computational systems that transfer data to a separate device. The new internal system allows the Thermal Cycling Spectrum to operate freely; thus, analyzing and computing data that is recorded throughout the cycling period. With the addition of a new internal system, a new port system has been created in order to withdraw and copy data that has been recorded within the internal drive. Two (2) new 3.0 USB ports and a mini-SD memory card slot have been included in order to provide multiple methods of transferring data from one device to another. The extra USB port is available in order to provide an external power source in order to run the Thermal Cycling Spectrum. This eliminates the problem of needing a stationary desktop computer in order to supply enough power to allow the device to function. Finally, the lid fastener has been drastically improved by including an alarm system that alerts the user when the lid has been securely fastened prior to the cycling period. One mini-LED screen will display a red screen until a green screen appears (on the other mini-LED screen), alerting the user that the lid has been properly fastened.

Fluorimeter Modifications



The fluorimeter will be made up of a cellphone docking station, a device that produces a blue LED light for each drop sampling, a rolling tray (with wheels) and a black box that protects any fraction of light from entering the premise of the fluorimeter setup. The cellphone docking station will feature a new way to dock your cellular device. By plugging in your cellular device, your cellphone will stay charged while performing during the length of the lab experiment. This will also securely fasten the device; thus, the distance between the drop sampling and the camera of the cellphone will not change. The docking station will have an external USB-cable that can plug into any computer to provide charging power. Next, the device that produces the blue LED light and holds the drop sampling will feature a rotational knob that raises or lowers the height of the drop sampling device. This will promote an equal level for the camera's perspective on the drop sampling; thus, an accurate side-view of the "beach ball" shaped drop sampling will be produced. The black box will not feature any modifications; however, the device that produces the blue LED light and the cellphone docking station will be placed on a moving tray. Before and after each trial of each drop sampling, the door for the black box will open upwards, like usual, and the tray will roll inwards/outwards of the black box. This will provide an easier and effective way of changing out each drop sampling without affecting the distance between the drop sampling and the camera of the cellphone; meanwhile, the black box will not have to be interfered with.


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 PCR for predicting the disease. 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 a Blackboard quiz. We are doing so for the sake of academic integrity and to curb any temptation to cheat.]


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