Name: Cyril Wassef - Design Strategy-Machine/Device Engineer
Name: Israel Brewer
Research & Development
Name: Manny Casildo - Research and Development - R&D Scientist
Name: Dale Caagbay
Role(s) Protocol and R&D
LAB 3 WRITE-UP
Original System: PCR Results
PCR Test Results
| Sample Name || Ave. INTDEN* || Calculated μg/mL || Conclusion (pos/neg)
| Positive Control || 7301649|| 62.48 || N/A
| Negative Control || 3786507.33|| 29.57|| N/A
| Tube Label: 1-1 Patient ID: 72537 rep 1 || 7457625.66 || 63.94 || pos
| Tube Label: 1-2 Patient ID: 72537 rep 2 || 9087739.66 || 79.2 || pos
| Tube Label: 1-3Patient ID: 72537 rep 3 || 4817516.33 || 39.23 || neg
| Tube Label: 2-1 Patient ID: 74083 rep 1 || 3558606.33 || 27.44 || neg
| Tube Label: 2-2 Patient ID: 74083 rep 2 || 4247969.66 || 33.89 || neg
| Tube Label:2-3 Patient ID: 74083 rep 3 || 4704281 || 38.17 || neg
* Ave. INTDEN = Average of ImageJ integrated density values from three Fluorimeter images
These following conditional statistics are based upon all of the DNA detection system results that were obtained in the PCR lab for 20 hypothetical patients who were diagnosed as either having cancer or not having cancer.
Bayes Theorem equation: P(A|B) = P(B|A) * P(A) / P(B)
Calculation 1: The probability that the sample actually has the cancer DNA sequence, given a positive diagnostic signal.
- A = Frequency of positive conclusions from cancer DNA sequence = 9/20 = .45
- B = Frequency of Total Positive DNA sequences = 26/60 = .433
- P (B|A) = Frequency of total positive DNA sequences given a positive conclusion = 25/26 = .962
- P(A|B) = .962
Calculation 3: The probability that the patient will develop cancer, given a cancer DNA sequence.
- A = Frequency of a mismatch for pos = 3/9 = .33
- B = Frequency of all mismatches = 5/9 = .55
- P (B|A) = Probability patient will get cancer = 3/10 = .3
- P(A|B) = .3
New System: Design Strategy
We concluded that a good system Must Have:
- [Simple software. This is of vital importance due to the fact it will attract more user. The easier the software is to navigate and work with, the better sales will be. It will also in turn help interpret data easier by displaying results on a simple scale.]
- [Access to imaging results in a hasty manner. Being able to interpret images quickly after interpretation is important so the users can justify what they are analyzing quickly and precisely. This in turn will help develop more in-depth analysis' by revealing faster results for more trials. This will increase validity of the information obtained in the long run.]
We concluded that we would Want a good system to have:
- [Ease of access. The group concluded that the easier the system is to access from the outside in, the more improvement the group and developers can make on a more frequent basis. That being said, these frequent improvements allowed by this aspect will only increase the use of the PCR machine. The group will be able to make improvements on the machine whenever it seems fit and necessary; possibly leading to newer discoveries more frequently.]
- [Low cost. Although it is not always possible to have a low cost because of the parts put into the system, the group leans towards the machine being affordable. These low costs will increase sales and provide itself with a better outlook on the environment. Its gain in reputation would help our group further sales in the industry and promote additional add-ons to this product if deemed fit. ]
We concluded that a good system Must Not Have:
- [A Troublesome USB connectivity. If this is a factor in production, the PCR would almost be coined as worthless due to the fact it needs to be attached to a computer with the correct software to even record the results. It would also ruin the company's reputation in the process by not appealing to buyers. It's a must to keep these machines in mass production and on the market.]
- [A hazardous casing. With the prototype being made of wood for the outside casing, it causes a fire hazard due to the fact it's highly flammable. Something more durable would be deemed fit to encase the material that is of such a large investment. This would give potential buyers a sense of assurance that the product is not simply something fragile and easily disposed of, but can hold its own.
We concluded that a good system Should Avoid:
- [Time-consuming amplifications. While the prototype PCR machine took just over an hour and a half to fully amplify and denature, the group intends on making the new product a bit more time efficient by reducing this time interval to just under an hour. Although its not a necessity like the others, its something the group wants to avoid to aid in productivity with the machine. ]
- [Inaccurate time read-outs. Although the time difference normally is usually within a few seconds, the difference in this time could ultimately affect the denaturing of proteins in the long run. We want to avoid this to avoid inaccurate readings of the proteins due to harm during the process. Accurate readings of the DNA from the machine are vital.]
New System: Machine/ Device Engineering
- The team planned on modifying the panels that encase the PCR system. By including shatter and scratch resistant Gorilla glass along with plexi glass for stability and glass for visibility, the system will seem more appealing to the user. It will also provide better stability for the system and do away with the potential fire hazard that was previously involved in the last design.
We chose to include these new features
- We changed the side panels of the PCR machine to gorilla glass, the bottom to plexiglass, and the front to just glass. This will ensure that the machine won't set on fire. This will also increase visibility of what is occurring during the machine from the glass. This will lead to more opportunities to see errors and improve them as the group concludes as necessary.
- First, the machine should be set up in such a way that the machine can be opened easily and accessed.
- Disassemble the machine with a screwdriver on the sides first
- Take each side and assemble each with appropriate glue to the sides; attaching to each other
- Let the machine sit and dry; approximately 2 hours at least optimally.
- Follow these instructions to assemble the PCR Machine
New System: Protocols
- We decided to change the materials of the PCR machine in order for it to be a safer machine.
Supplied in Kit
- Reaction Buffer
- DNA polymerase
- Sample DNA
- SYBR Green Dye
We chose keep the protocols the same as the original system
- Feature 1 - The PCR machine effectively amplifies the DNA with little error.
- Feature 2 - The software used in the PCR testing is very easy to use.
- Feature 3 - The fluorimeter gave us a good idea whether a sample was positive or negative
DNA Measurement and Analysis Protocol
- Step 1: Gather materials and safety equipment
- Step 2: Put fluorimeter in dark box, keep one hatch open for ease of access
- Step 3: Level the phone camera lens with fluorimeter
- Step 4: Calibrate fluorimeter with water
- Step 5: Dilute samples with with appropriate solution (80ug)
- Step 6: Place 80ug of solution onto fluorimeter slide (make sure blue light runs through drop)
- Step 7: Run fluorimeter again
- Step 8: Take picture and repeat with the different samples
- Step 9: Open image J
- Step 10: Split picture into three channels and choose green
- Step 10: Create a circle around the droplet in picture
- Step 11: FInd the aveintdent for each droplet by clicking analyze, then measure
| Calf Thymus DNA solution concentration (microg/mL) || Volume of the 2X DNA solution (uL) || Volume of the SYBR GREEN I Dye Solution (uL) || Final DNA concentration in PicoGreen Assay (ng/mL)
| 0 || 80 || 80 || blank
| .25 || 80 || 80 || .125
| .5 || 80 || 80 || .25
| 1 || 80 || 80 || .5
| 2 || 80 || 80 || 1
| 5 || 80 || 80 || 2.5
New System: Research and Development
- In summary the CHEK2 gene, short for Checkpoint Kinase 2, encodes the protein that inhibits the CSC25C phosphatase when activated. In other words, it helps to slow down cell growth and division. This particular phosphatase helps to "counter" the tumor cells and bring in a better chance of survival for damaged and affected cells. It slows down the tumor cell's rapid growth rate and allow medications to work on affect areas. Associating with this gene, SNP, short for 'Single Nucleotide Polymorphism', is clinically significant in that it is a pathogenic substance. In clarification, it shows if cancer is evident in cells or not. Because of it's pathogenic properties, it flips the function of CHEK2 in reverse and promotes cancer cell growth.
In the worksheet that we've filled out, we found that the sequence for CHEK2 is ATT. If it is altered in anyway, we can come to conclusions that there may be the presence of a carcinogen in cells. We can verify this by the mutation from ATT to ACT.
- The PCR machine will include nucleotides that will bind to certain base pairs and promote cancer cell growth. This is very helpful in a visual sense, obviously if the patient has cancer then cancer cells will grow to promote visibility of the cells and accuracy of the PCR machine.
Primers for PCR
- The primers that were selected was: Forward primer: Reverse Primer:
- A disease allele will give a PCR product because the primers that were selected are only going to match the DNA sequence that is cancerous. When the primer binds to a cancerous allele it will then start to grow thus visible if cancerous. However there will not be a PCR product if there is a non disease allele for the same reason. When the primer and the DNA are put together there will not be any binding because the nucleotides will not match correctly onto the DNA thus negating any replication and any visibility or product.