BME100 s2014:T 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

Image:PCReate1.PNG


Group Members:

Name: Caitlin R. Byrne
Name: Caitlin R. Byrne
Name: Megan McGuire
Name: Megan McGuire
Name: Wade Savage
Name: Wade Savage
Name: Theo Hall
Name: Theo Hall


PCReate

"An easier way to divide and conquer."

  • Our product has designed a new type of PCR intended for a faster and more accurate reaction.



LAB 6 WRITE-UP

Computer-Aided Design

TinkerCAD

TinkerCAD is a free and simple online tool, where the user can design a 3D model that is automatically 3D Printer compatable. In class, all members of the group were required to register. Two members of our group was required to take at least seven lessons on how to use TinkerCAD and all of it's tools. Then we were then given the task of looking at the OpenPCR machine and improving upon it to make it better. After analyzing the positives and negatives of each step in the PCR reaction process and analysis, we then designed our improvements on TinkerCAD.

Our Design

Image:PCReateSystem.PNG


PCReate, after analyzing the current process of a polymerase chain reaction using the OpenPCR machine, decided to make slight changes upon the machine itself. We decided to add a lid on top the the heating structure to be a standard height. This way, there is no adjustment of the top heating plate. This provides a complete heating of the test tubes placed inside the PCR machine. This is ultimately different then the old PCR system, by eliminating the human error of lowering the heating plate. This caused a risk for error, because if the lid was tightened to tight, the lid would open and the PCR machine would not happen, or if the lid was tightened too loose the top of the test tube would not be heated fully. Because of the loss of heat, the PCR reaction would not complete to full capacity. PCReate decided to eliminate this human error in order to result in a more complete polymerase chain reaction.


Feature 1: Disease SNP-Specific Primers

Background on the disease-associated mutation


Nucleotides are compounds consisting of a nucleotide linked to a phosphate group to form the basic structural unit of DNA. Polymorphism is a term used when two or more specific different phenotypes exist in the same population of a species. A DNA sequence, made up of the nucleotides A, T, C, and G (adenine, thymine, cytosine, and guanine) in the same genome differ between species. Some of these genetic variations are short and made up of single nucleotide polymorphisms (SNPs). After researching the SNP rs237025, it was found to be a variation in Homo sapien (human) species. This variation is found in chromosome 6, and has been linked to health-related illness diabetes. The gene SUM04 stands for small ubiquitin-like modifier 4. These are modifiers that are attached to proteins and control the target proteins' subcellular localization, stability, or activity. Allele's are one of two or more alternative forms of a gene that arises by mutation and are found at the same place on a chromosome. The disease-associated allele rs237025 contains A-T-G, different from the non-disease alleles are 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'


How the primers work:


A primer is a strand of nucleic acids that serve as a starting point for DNA synthesis during a PCR reaction. The non-disease allele will not bind to the PCR reaction template. Forward primers bind to disease templates while reverse primers will perform the same function to the complimentary templates. Because this allele does not bind 100% to the PCR, the raction will not occur and there should be no result from this allele.



Feature 2: Consumables Kit

The components included in the kit include:

- Reagents:
   - PCR mix
   - Calf thymus DNA 
   - Primer solution
   - SYBR Green solution
   - buffer
- Plastic tubes
- Glass slides
- Pipettor and disposable tips
- Biohazard disposal kit
- Sharps container

Our consumables will be packaged inside a container with separate sections for the reagents, plastic tubes, pipettor, and glass slides, etc. which differs from the original, less organized design. All components will be labeled. This will allow for easy identification of each part of the fluorimeter. All items will be packaged securely within the container so that the components, specifically the glassware, will be in a safe environment. The SYBR green solution, which is sensitive to light, will be packaged in its own container in order to protect it from light and provide easy access to the solution when it needs to be used. This is a better alternative to the flimsy aluminum foil which was unable to adequately cover the solution. By changing this design flaw, we can minimize error in the results.

Feature 3: Hardware - PCR Machine & Fluorimeter

PCReate's New System Includes

The PCReate system will include one (1) consumables kit, one (1) modified and updated OpenPCR machine, and one (1) flourimeter. Consumables regarding the primers and PCR reaction mix will be measured out ahead of time by PCReate, for a more percise measurement. Assembly will still be required for the OpenPCR machine and flourimeter if the customer does not want the machine preassembled. If selected, PCReate will assemble it in the warehouse; but an extra fee will be charged for assembly and a more expensive shipping. With all three of these components, you will recieve the entire PCReate system.


PCR Machine Modifications

PCReate identified the main problem with the OpenPCR machine had to do with the heating system. When put together correctly, the machinery inside worked fine. What we found to be the problem was the heating system to conduct the PCR reaction. On top of the OpenPCR machine is a box, that contains the heating plates and a spot to place test tubes. We wanted to keep the same size and number of test tubes inside the heating compartment, however we wanted to get rid of the "tightening system" that lowers the top heating plate over the top of the test tubes once the lid is closed. In order to get the most accurate reaction, this top heating plate must be aligned perfectly. Being human, this is not garunteed to happen. The person who conducts the experiment either tightens the plate of the OpenPCR machine too tight and pops open the lid, or the heating place is too far away from the test tubes and they don't heat throughly. With the PCReate system, we have designed the lid of the OpenPCR machine to be already adjusted to the generic test tube size given to you as a consumable with an order of the PCReate OpenPCR system. This garuntees and even reaction throughout the entire test tubes and eliminates chances for error.

Flourimeter Modifications

PCReate did not modify the design to the basic cell phone flourimeter, but plan to in the near future. For now, the basic flourimeter is given to the consumer with clear and concise instructions.


Bonus: Tests related to Bayesian

In the calculations 3 and 4 the reliability was very low. With our given test group there was much error within the processes of the machine. The rate of success for accurate reading was well below 95%. Accurate reading being a positive test with positive signs. There were crossovers in the readings, meaning a negative result came with positive markers and vice-verso. We're sure that in a larger group of subjects the error might have been less but with these readings the PCR machine doesn't seem to be reliable.


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