BME100 f2013:W900 Group10 L6

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'''PCR Pros
'''PCR Pros'''

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BME 100 Fall 2013 Home
Lab Write-Up 1 | Lab Write-Up 2 | Lab Write-Up 3
Lab Write-Up 4 | Lab Write-Up 5 | Lab Write-Up 6
Course Logistics For Instructors
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Name: Barrett Anderies
Name: Barrett Anderies
Name: Joslin Jose
Name: Joslin Jose
Name: Liam Williams
Name: Liam Williams
Name: Duran Charles
Name: Duran Charles

PCR Pros


Computer-Aided Design


We used the 3D CAD software TinkerCAD to modify the given PCR tube CAD file. We added linking structural members between all of the PCR tubes to make them into a single unit. This makes them more convenient to fill and transport. Furthermore, we modified the directions the lids open. This minimizes the area the tube array takes up when the lids are open.

Implications of Using TinkerCAD for Design

CAD software, like TinkerCAD, is essential for rapid product design and prototyping. An example of this would be modifying the OpenPCR machine to hold 36 tubes rather than just 16. This would require a different PCR block that would take up more space than the original block. With CAD software, we can design a new PCR block and corresponding heating element and add it to the current CAD model of the OpenPCR machine. This way we can check to see if our design is viable and will fit in the current machine, or what other modifications we will need to make to accommodate our additions. After confirming that our design is viable, we can 3D print the parts we modified and add them to a current OpenPCR machine to practice assembly and display our design to interested parties (investors, etc.) without actually going into production.

Feature 1: Cancer SNP-Specific Primers

Background on the cancer-associated mutation

DNA, deoxyribonucleic acid, is a polymer composed of the nucleotides (monomers) Adenine, Guanine, Cytosine and Thymine. The arrangement of these nucleotides in DNA encodes the genetic instructions that facilitates the development and operation of all organisms on earth. Since an organism is dependent upon its DNA for proper functioning, mutations in DNA can potentially have serious consequences. An example of a genetic mutation is a single nucleotide polymorphism, or SNP, where a single nucleotide is replaced by another (and as a result the corresponding nucleotide on the the complementary DNA strand is also replaced). For example an Adenine is replaced by Cytosine (in which case the corresponding nucleotide on the other strand would go from Thymine to Guanine). In many cases an SNP will not cause any significant change in an organism's phenotype, but one instance where this does occur is an SNP of rs17879961 in chromosome 22 in humans. rs17879961 is in a tumor suppressor gene that produces a protein called CHEK2 (checkpoint kinase 2) which helps regulate cell mitosis. When an SNP occurs at rs17879961 CHEK2 does not operate correctly. This greatly increases the risk of cancer in an affected individual.

Primer design

  • Forward Primer: 3'-ACTCACTTAAACCATATTCT-5'
  • Cancer-specific Reverse Primer: 5'-GGTCCTAAAAACTCTTACAC-3'

A DNA primer is a short strand of nucleotides (usually about 20 nucleotides) that is complementary to a region on a template DNA strand. Because DNA will naturally bind to complementary strands, the primer attaches itself to the region on the DNA strand to which it is complementary. In general, a 20 nucleotide-long DNA strand will only match a single region (or no region) on a DNA strand, and will thus consistently bind only to that region. However, primers that are less than 20 nucleotides long run the risk of matching more than one sequence on the template DNA strand. This is important because we use primers to "block out" a region on the DNA strand we wish to amplify. Because a primer can only bind to a template DNA strand that contains its exact complement, we can take advantage of this in PCR when we are looking for an SNP. If we create a primer that is the complement of a region of DNA with a known SNP, the primer will ONLY BIND to a template strand that contains this SNP. Furthermore, because PCR cannot function unless primers attach to the DNA template strands, we can use this to determine if an SNP is present in a given template strand. If a PCR reaction containing a template DNA strand and a primer with the complement of a cancer-associated SNP results in a large (exponential) amplification of DNA, we can conclude that this template strand contains the cancer-associated SNP. If, however, there is little or no amplification we can conclude that our primers did not attach properly, indicated that the cancer-associated SNP is not present on our template strand.

Feature 2: Consumables Kit

In our kit, the consumables will consist of tubes containing the PCR reagents, tubes containing the SYBR Green I dye, the micropipette tips (to be used with the included micropipette) and safety equipment (gloves and eyewear). To ensure that users will be able to easily distinguish which tubes contain what, the tubes will have black markings on the lid to indicate contents as well as be color coordinated with their contents. Furthermore, SYBR Green I dye is prone to bleaching in the presence of ultraviolet light. Therefore, the tubes containing the SYBR Green I dye will be made out of a plastic that blocks ultraviolet light. Besides these modifications, the tubes will be the same as current PCR tubes and will be packaged as a unit (all of the tubes are connected by a small bar, and can be broken down into smaller units). Current micropipette and tip systems work well, so our kits will contain commercially available micropipettes and micropipette tips. The tips will be packaged in trays that stack on top of each other with the tips of trays on top partially inserting into the tips below. This conserves space and allows the tip tray assemblies to be packaged as a single unit. In addition to the above mentioned packaging, some consumables will have a resealable bag to safely store unused materials.

Feature 3: PCR Machine Hardware

Our kit includes a PCR machine similar to the OpenPCR machine. However, unlike the OpenPCR machine, ours comes assembled so it is ready to use out of the box. Furthermore, our PCR machine is constructed out of durable, heat resistant plastic and does not need a computer to operate. It has buttons on the LCD screen module that allows for stand-alone programming and operation. Our machine also has increased capacity, able to hold 36 PCR tubes at once, and has a heating lid that opens towards the back of the machine so that users do not have to work with liquids over the LCD screen. This makes it less likely that the LCD screen and related components will be damaged by spills.

Feature 4: Fluorimeter Hardware

Our kit includes a fluorimeter designed to be used in conjunction with a smartphone to analyze PCR product samples. Our design incorporates the reliable, single-slide, single-light design of other fluorimeters, but tackles the problem of camera image inconsistency by using a camera tray that is adjustable in two directions (higher-lower, closer-further). This way, almost any smartphone can be accurately positioned and held steady for the duration of the experiment.

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

  • Calculation 3 detects the probability CHEK2 PCR will correctly diagnose a positive results for a person who indeed has cancer. The calculation in the data collected resulted in a number that was low and less than one indicating that the CHECK 2 PCR sensitivity (ability to predict a true positive result) is not very good.
  • Calculation 4 describes the probability CHEK2 PCR will correctly diagnose a negative result for a patient who does not have cancer. The results of the specificity (ability to predict a true negative)was a grader than one. Therefore, indicating CHEK2 PCR is accurate a diagnosing a negative result for patients who do not have cancer.
  • Overall, the Bayesian stats implies that CHEK2 PCR is not a reliable diagnostic device because it has a high probability of giving a patient a negative result when the patient does indeed have cancer.
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