Name: Manny Casildo - Research and Development - R&D Scientist
Name: Student Role(s)
Name: Student Role(s)
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
Bayesian Statistics
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.
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
SYSTEM DESIGN
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.
KEY FEATURES
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.
INSTRUCTIONS
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
DESIGN
MATERIALS
Supplied in Kit
SYBR Green - 60 ug
DNA Samples
Fluorimeter
Phone
Micropipettes
PROTOCOLS
PCR Protocol
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 SYBR green
Step 6: Run fluorimeter again
Step 7: Take picture and repeat with the different samples
Step 8: Open image J
Step 9: Create a circle around the droplet in picture
Step 10: FInd the aveintdent for each droplet by clicking analyze, then measure
New System: Research and Development
BACKGROUND
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. specification 1 - explanation of how an aspect of the primers addresses any of the specifications in the "New System: Design Strategy" section
Design specification 2 - explanation of how an aspect of the primers addresses any of the specifications in the "New System: Design Strategy" section
Etc.
DESIGN
Primers for PCR
Our primers address the following design needs
Design specification 1 - explanation of how an aspect of the primers addresses any of the specifications in the "New System: Design Strategy" section
Design specification 2 - explanation of how an aspect of the primers addresses any of the specifications in the "New System: Design Strategy" section
Etc.
New System: Software
[THIS SECTION IS OPTIONAL. If your team has creative ideas for new software, and new software is a key component included in your new protocols, R&D, or machine design, you may describe it here. You will not receive bonus points, but a solid effort may raise your overall page layout points. If you decide not to propose new software, please delete this entire section, including the ==New System: Software== header.]
BME 103 Spring 2013
