BME100 s2014:T Group10 L6

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The Group 10s of Tomorrow

Sean-Christopher Bradbury
Thursday Group 10 Member
Suyen Go
Thursday Group 10 Member
Nathaniel Kirkpatrick
Thursday Group 10 Member
Maya Robinson
Thursday Group 10 Member
Lissette Valle
Thursday Group 10 Member

The Group 10s of Tomorrow presents:
OpenerPCR Deluxe


LAB 6 WRITE-UP

Computer-Aided Design

TinkerCAD

TinkerCAD is a 3D imaging software used to create three-dimensional models and designs with ease. Those models can then be printed using a 3D printer. It has pre-built shapes that a person can utilize in combination with other shapes to create any 3D design they can think of. Also, there is an active design community where objects built by others are shared and built upon to make computer aided design more collaborative and easy.


Our Design


OpenerPCR Deluxe now with twice the capacity! OpenerPCR Duluxe with rotated double-wide heated lid! OpenerPCR Deluxe with integrated whiteboard grid system.

At The Group 10s of Tomorrow (Ltd.) we strive for excellence, efficiency and extreme people-helping. Accordingly, we at G10T (Ltd.) have taken it upon ourselves to solve a problem facing those who rely on a system of PCR and Fluorimetry to diagnose patients with various SNPs. Traditionally, OpenPCR machines only have 16 spaces for PCR tubes. This means only four patients with three replicates each (along with a positive and negative control) can be thermocycled at one time. This bottleneck in efficiency increases costs, reduces productivity, increases costs, and most importantly prevents patients from getting quick and accurate diagnoses.

In this landscape of inefficiency we humbly introduce OpenerPCR Deluxe. Its state-of-the art PCR tube system allows for 32 PCR tubes to be thermocycled at one time. This allows for 10 patients with three replicates each, along with one positive and one negative control, to be processed for fluorimetry at once. By increasing the patient capacity by 250%, we alleviated the troublesome bottleneck inherent in the original OpenPCR design.

The educated researcher may ask, "but how will I keep all of those PCR tubes straight?" and they ask a valid question. 16 tubes are hard enough to keep track of, especially when the labels are sometimes removed during thermocycling. How would a researcher be expected to keep tabs on twice that number of tubes? With OpenerPCR Deluxe the researcher does not have to! We have integrated state-of-the-art Whiteboard technology to allow for easy sample labeling.


Feature 1: Disease SNP-Specific Primers

The disease for the SNP-specific primers derives from the variation of single nucleotide polymorphisms that varies throughout a variation of species. Through this source of genetic specification scientists are able to detect a single has pair mutation simply from a specific locus. Since the loci of a humans genetic code has been conserved during evolution this can serve as a quantitative trait analysis when evaluation the affirmation of a certain disease.


rs237025 is a short genetic variation found in Homo sapiens located on chromosome 6. This SNP is associated with the genes SUMO4 (387082) and TAB2 (23118). SUM04 stands for Small Ubiquitin-like Modifier 4 which leads to negative regulation of NF-kappa-B-dependent transcription of the IL12B gene. There are many diseases associated with this SNP. This SNP is associated with Type 1 Diabetes, Type 2 Diabetes and Vogt-Koyanagi-Harada disease. The disease-associated allele has the sequence ATG at numerical position 149721690.


Primer design

  • Disease SNP-specific Forward Primer: 5'-TGAACCACGGGGATTGTCAA-3'
  • Reverse Primer: 5'-TGTGGTGGAACCAAATTGCA-3'

How the primers work: Primers are disease-sequence specific because they require perfect bonding for replication to occur. Since the disease allele is at the beginning, the forward primer will not bind to the non-disease-associated sequence at all because the genetic code required for bonding is not present. However, the reverse primer will bind to both the disease-associated sequence and the non-disease-associated sequence because they both have the same genetic sequence that far away from the disease allele. Fortunately though, since the replication process is repeated multiple times, the few copies of the non-disease associated SNP the reverse primer produces will be drowned out by the exponentially increasing disease-associated sequence copies. Each non-disease associated SNP will be copied one time for each cycle, while each disease-associated SNP will be copied twice (once from the forward primer, once from the reverse primer).


Feature 2: Consumables Kit

The consumables in the improved PCR machine will consist increasing the amount of samples which will elad to also increasing the amount of consumables needed in the process. This will not only make amount of samples larger retains essential data such as negative and positive controls. The PCR machine will consist of:

  • PCR Tubes(32)
  • PCR Reaction Mix(32 with 50 μL each)
  • Template DNA with Primer(32)
    • Positive Control(1)
    • Negative Control(1)
    • Patient 1 DNA with primer (3 replicates)
    • Patient 2 DNA with primer (3 replicates)
    • Patient 3 DNA with primer (3 replicates)
    • Patient 4 DNA with primer (3 replicates)
    • Patient 5 DNA with primer (3 replicates)
    • Patient 6 DNA with primer (3 replicates)
    • Patient 7 DNA with primer (3 replicates)
    • Patient 8 DNA with primer (3 replicates)
    • Patient 9 DNA with primer (3 replicates)
    • Patient 10 DNA with primer (3 replicates)
  • 200μL Micropipeter
  • Disposable Tips

The weakness of doubling the tubes that will be tested at one time will be keeping track of which tube is which because there are so many tubes that have to be accounted for. In the midst of tracking the data, tubes will be easily confused and possibly identified as a wrong subject or replicate.The packaging plan addresses weaknesses confusion of the tubes in the rack. Our tube rack will contain clear labels identifying the patients and the replicates.



Feature 3: Hardware - PCR Machine & Fluorimeter

The PCR machine will be used to amplify the genetic sequence in question. These samples will then be subjected to single-drop fluorimetry with a photo-reactive chemical called SYBR Green I that will glow green when bound to the target SNP sequence.

Our PCR machine features twice the PCR tube capacity. Therefore, the OpenerPCR Deluxe is able to test two-and-a-half times as many patients compared to the original version. Instead of only four patients with three replicates each along with a positive and negative control being tested at once, the OpenerPCR allows for 10 patients to be tested at once. Furthermore, due to our larger batch size, positive and negative controls only need to be run once per 10 subjects, rather than once per 4 subjects as before. This allows for more time and resources to be spent on testing the actual subjects rather than the positive and negative controls.

Our PCR Tube holder is twice the length of the traditional square 4x4 OpenPCR design. To accommodate this increase, the heated lid's length was doubled as well and its hinges were rotated to allow for easier access. To confront the problem of keeping track of the numerous PCR tubes, the OpenerPCR Deluxe has an entire side of its body made out of cutting-edge, state-of-the-art whiteboard material with a pre-printed grid system, making labels more convenient and removed from to the precarious conditions of the thermocycling environment. (Dry-Erase marker sold separately)



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


With the probability of a patient developing the disease given a positive final test conclusion being over one half, it can be reasonably assumed that more than half of the people that test positive will develop diabetes. While these results do not seem incredibly predictive, the the true ability of the test lies in the probability of a patient not developing diabetes given a negative final test conclusion. This probability is close to one, meaning that almost 100% of the people that test negative for the disease SNP will develop diabetes. This means that our test has high sensitivity. The test errs on the side of false positives. It is almost certain that those with negative test results will not develop diabetes, but only more than half of the people that test positive for the disease SNP will develop diabetes. Accordingly, our test has low specificity.

Works Cited

Harbron, Rapley S. "SNP Genotyping." Wikipedia. Wikimedia Foundation, 18 Apr. 2014. Web. 21 Apr. 2014.