BME103:T930 Group 15

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Lab Write-Up 1
Lab Write-Up 2
Lab Write-Up 3
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Contents

OUR TEAM

Name: Suhail HiermandiRole: Research and Development, Experiment Protocol
Name: Suhail Hiermandi
Role: Research and Development, Experiment Protocol
Name: Andrew TrujilloRole: Research and Development
Name: Andrew Trujillo
Role: Research and Development
Name: Elias MloukhiyehRole: Open PCR Machine Tester
Name: Elias Mloukhiyeh
Role: Open PCR Machine Tester
Name: Emma GodderyRole: Experiment Protocol Planner
Name: Emma Goddery
Role: Experiment Protocol Planner

LAB 1 WRITE-UP

Initial Machine Testing

The Original Design
Image:PCR Machine Image.png
An Open PCR Machine is a device that is used to learn if a sample of DNA reacts with a select gene. It does this by fluctuating the temperature of the samples. With the temperature fluctuating, strands of DNA break apart and the chemical reacts withe the single strand of DNA by completing the strand.


Experimenting With the Connections

When we unplugged the LCD display from the circuit board, the machine LED display powered down and did not display anything.

When we unplugged the white wire that connects the circuit board to the heat sink, the temperature reading on the LED display screen showed the incorrect temperature.

Test Run

During the "Test Run" portion of the lab on the 25th of October 2012, the OpenPCR machine did as it was supposed to. It fluctuated the heat of the test samples and it also had the same readings on the computer program as well as the LED display screen of the OpernPCR machine.




Protocols

Polymerase Chain Reaction

PCR is a way to copy a segment of DNA in order to analyze that DNA successfully. First, DNA is extracted from a cell. It does not have to be a lot because the PCR will amplify it. This DNA will be added to a tube with primers, nucleotides and Taq Polymerase. This is heated, cooled, then heated because the temperature changes will help unwind the DNA, make the primers work, and bind the new DNA strands. By running this cycle many times, many copies of DNA can be created.

How to amplify a patient's DNA sample:
1. Put extracted DNA into a PCR tube.
2. Add primers to the PCR tube.
3. Add nucleotides to the PCR tube.
4. Add DNA Taq Polymerase to the PCR tube.
5. Place this DNA tube into DNA Thermal Cycler.
6. Thermal cycler heats to 95 degrees Celsius for 30 seconds.
7. Thermal cycler cools to 57 degrees Celsius for 30 seconds.
8. Thermal cycler heats to 72 degrees Celsius for 30 seconds.
9. These cycles happen 30 times.

PCR Master Mix Components:
GoTaq® Colorless Master Mix, 2X 25μl
upstream primer 10μM
downstream primer 10μM
DNA template 1–5μl
Nuclease-Free Water to 50μl

Reagent Volume
Template DNA (20ng) .2 μl
10 uM forward primer 1.0 μl
10 uM reverse primer 1.0 μl
GoTaq master mix 50.0 μl
dH2O 47.8 μl
Total Volume 100.0 μl



Samples we tested:
Positive control: cancer DNA template (+)
Negative Control: no DNA template (-)
Patient 1: ID 12329(*) Replicate: 1 Label: P1R1
Patient 1: ID 12329 Replicate: 2 Label: P1R2
Patient 1: ID 12329 Replicate: 3 Label: P1R3
Patient 2: ID 61058(**) Replicate: 1 Label: P2R1
Patient 2: ID 61058 Replicate: 2 Label: P2R2
Patient 2: ID 61058 Replicate: 3 Label: P2R3
*Patient 1: Female, 61 years of age.
**Patient 2: Male, 57 years of age.


Flourimeter Protocol
Steps to set up the Flourimeter:
1. Obtain a glass slide specially designed for flourimeter procedure.
2. Place the slide on the device with the light and line up the light so that it is shining between two of the circles.
3. Turn the box upside down and un-buckle one of the sides so that it can be moved up and down.
4. Put the device with the blue light under the box in the back and place the phone stand in front of it.
5. Put a phone in the phone stand (iPhone 4 was used in this lab) and move the stand close enough to take a picture.
6. Place a drop of SYBR Green on one of the circles and a drop of whatever is being tested on the circle behind it. They should merge to be one big droplet.
7. Close the box as close to all the way as possible and take a picture using the phone with no flash.
8. Record image number and sample type.

Example:

Materials


Set up with camera


Closing the lid

Image J Procedure:
1. Take a picture of the flourimeter assembly using an iPhone 4.
2. Send the picture to a computer with Image J software.
3. Open the file with Image J software.
4. Use the menu selection analyze > set measurements and chose Area Integrated Density and Mean Grey Value.
5. Use this menu selection Image > Color > Split Channels
6. Since SYBR Green I fluoresces green you will use the file that has green next to it.
7. Activate the oval selection and draw an oval around the green drop image and then select analyze > measure. Write down the sample number and the numbers measured.
8. Get a background reading by drawing another oval of the same size in the green file for the background above the drop to get the "noise".
9. Select analyze > measure and write down the sample number and numbers measured and label this as background. Save the measurements to an excel file.

Research and Development

Specific Cancer Marker Detection - The Underlying Technology

The Open PCR machine works by heating a sample of DNA to 95 degrees Celsius to separate the sample into single strands. Then, the machine cools to 57 degrees Celsius so that primers can attach to the strand to serve as a probe to a matching sequence. The primer that was used in our experiment was r17879961, a cancer-associated sequence, that will detect if the DNA sample tested positive for cancer. Then, the machine heats up to 72 degrees Celsius where MgCl12 attaches to Taq enzyme, so that Taq can take the free floating dioxynucleotide triphosphates and attach them to the stand to form two new separate and similar DNA strands. Now if the process produce new strands, then the DNA tested positive. If none were present, the DNA tested negative. The reason behind this is that r17879961 only can bind to a sequence that matches its own on the single stranded DNA. This sequence is 5’-AAACTCTTACACTCCATACAT-3’. The cancer mutation site is located at the 12 base pair, 5’-ACT-3’. If the base pair is a C, it is positive for cancer mutation. The base pair should truly be a T, making the sequence 5’-ATT-3’. The particular cancer that is associated with the r17879961 primer is colon rectal cancer. The information regarding this particular cancer sequence can be found on the National Center for Biotechnology Information, in the file “.0002 LI- Faurmen Syndrome”. A study done in the file tested the C mutation r17879961. The results that were found were that 7.8% of C mutations were found in cancer patients, and that 5.3% of C mutations were in Finland. The results then can show that 7.8% of the cancer patients had a positive test, and of that 7.8%, 5.3% are in Finland.




Bayes Rule is used to test all data collected and find the limitations of the data. It can be used to fin the positive predictive value (PPV) and the negative predictive value (NPV). The variables used are: C= Cancer Present T= Positive Test P(A|B)= Probability of A, given B

~= not

These vaiables can find the prior probability and conditional probability. Prior Probability: p(C) {probability of cancer} Conditional Probability: p(T|C) {probability of a positive test} p(T|~C) {probability of a negative test} When the information is found, Baye’s Theorem can be utilized: p(C|T)=[p(T|C)*p(C)]/[p(T|C)*p(C)+p(T|~C)*p(~C)] Baye’s Reasoning can also be used to find clinical diagnostic specifics; sensitivity, specificity, PPV and NPV. Sensitivity= p(T|C) x 100% Specificity= [1-p(T|~C)] x 100% PPV= p(C|T) x 100% NVP= [1-p(C|T)] x 100%

Results


Water


Calf Thymus DNA

Sample Integrated Density DNA μg/mL Conclusion
PCR: Negative Control (Water) 2089225 1.52 positive
PCR: Positive Control (Calf Thymus) 714695 0.5212 no signal (negative)
PCR: Patient 1 ID 12329, rep 1 4108573 2.996 positive
PCR: Patient 1 ID 12329, rep 2 3003328 2.19 positive
PCR: Patient 1 ID 12329, rep 3 2139854 1.5606 positive
PCR: Patient 2 ID 61058, rep 1 762689 0.556 no signal (negative)
PCR: Patient 2 ID 61058, rep 2 1035447 0.7551 no signal (negative)
PCR: Patient 2 ID 61058, rep 3 535664 0.3906 no signal (negative)


KEY

  • Sample = subject DNA that is contained in one test tube
  • Integrated Density = Integrated density is the of all values of the pixels in the image
  • DNA μg/mL = The DNA concentration was measured in micrograms/mL. We found the DNA concentration by multiplying each of the Integrated Densities by 2, then dividing the Integrated Density by the calf thymus DNA value. That gives us our DNA concentration.
  • Conclusion = We concluded that if the DNA concentration was higher than 1.0 micrograms/mL, then it was positive for the cancer gene and if it was less than 1.0 it showed no signal and lacked the cancer gene. For example, our calf thymus DNA had a 2.0 DNA concentration so it was positive for the cancer, while the water which didn't have a DNA concentration didn't show any signs of cancer


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