Name: Jocelynn Christensen
Role: Experimental Protocol Planner
Name: Sam Zimmerman
Role: OpenPCR Machine Engineer
Name: Ryan Uchimura
Role: Experimental Protocol Planner
Name: Dakota Styck
Role: OpenPCR Machine Engineer
LAB 1 WRITE-UP
Initial Machine Testing
The Original Design
The images above depict our OpenPCR machine. It performs polymerase chain reactions (PCR), a process in which a particular DNA sequence is amplified. The DNA sequence can then be easily analyzed. The OpenPCR machine accomplishes the amplification of a DNA sequence through a series of heating and cooling sequences.
Experimenting With the Connections
When we unplugged the LCD screen from the circuit board, the machine stopped displaying information on the LCD screen.
When we unplugged the white wire that connects the circuit board to the heating block, the heating block would not heat up.
We first tested our OpenPCR machine (machine #6) on October 25, 2012. We ran the machine through a preprogrammed sample test run. At first we were not getting consistent temperature readings outputting to the LCD screen; however, after we rebooted the OpenPCR machine and ran the test again the machine worked perfectly.
Polymerase Chain Reaction
1. The Polymerase Chain Reaction or PCR works by singling out a single piece of DNA and then multiplying it so there's millions of copies of one strand of DNA. It's a step by step process that first occurs by heating up the DNA to 100°C in order to denature the hydrogen bonds between the two strands of DNA so that both sides of the DNA can be accesible to copy. After the strands are separated, specific primers are added to locate the section of DNA to be amplified. Next, the Taq DNA polymerase is added which actually copies the section of DNA desired and synthesizes the second half of each strand. After this there are only a few copies of the DNA which is why the machine then replicates more strands by repeating the process multiple times until there are millions of copies.
- 1. Collect DNA from patients
- 2. Mix the DNA samples with the GoTaq master mix and place into the PCR machine
- 3. Heat Denaturation- the sample is heated to break the bonds between the strands
- 4. Primer Annealing- the solution cools and the DNA matches up to the primer.
- 5. Extension- The DNA replicates to produce millions of copies of the one strand of DNA .
3. The GoTaq master mix contains 400µM dATP, 400µM dGTP, 400µM dCTP, 400µM dTTP, and 3mM MgCl2
4.Reagents used in our PCR test
| Reagent || Volume
| Template DNA (20ng) || 0.2μL
| 10μM forward primer || 1.0μL
| 10μM reverse primer || 1.0μL
| GoTaq master mix || 50.0μL
| dH2O || 47.8μL
| Total Volume || 100.0μL
5. 8 Samples
| Positive Control
||Sample 1 ID:19185
||Sample 2 ID:19185
||Sample 3 ID:19185
||Sample 1 ID:88142
||Sample 2 ID:88142
||Sample 3 ID:88142
Out of the eight samples we ran the PCR on, one was a positive control with the cancer DNA template, the other was a Negative control with no cancer DNA template. Then we had three samples of a 57 year old male's DNA (patient ID 19185). We also had three samples of a 63 year old female's DNA (patient ID 88142).
1. Take the file box and place it upside down (after it's been emptied) so that there is now an area that will restrict as much light as possible from coming through in the pictures.
2. Place the hydrophobic slide in the center of the fluorimeter and align it so that the row of dots is directly in the middle of the blue lazer.
3. Using a pipet, place two droplets of water gently on the middle hole of the slide.
4. Next use the pipet to add two droplets of the PCR solution for the first sample with the two water droplets. This must be done cautiously so that the droplets will stay in the center and adhere to each other properly.
5. Turn on the light source on and place the fluorimeter as far back as possible inside the upside down box.
6. Turn the camera on a smart phone and adjust the settings accordingly. Inactivate the flash, set iso to 800 (or higher if possible), set white balance to auto, exposure to the highest setting, saturation to highest setting and contrast to the lowest setting.
7. Place a smartphone camera into the cradle and then move the cradle in front of the fluorimeter at the perfect distance for good resolution (may take some adjusting).
8. Take a picture of the mixture on the fluorimeter (best done if camera is on a timer so that you can fully close the box and avoid excess light exposure.
9. Repeat steps 2-8 for each sample.
IMAGE J Analysis Instructions:
1. Download the Image J software
2. Open Image J
3. To chose your pictures click 'file' and 'open' then select the image you wish to analyze
3. Once your image is opened highlight and click on "analyze", "set measurements", and check the boxes "area" "integrated density" and "mean gray value" in order to just analyze the pieces for this lab.
4. After this you have to highlight and click "image", "color", then click "split channels"
5. This splits the image you have into three new images. For this assignment just keep the image "green" and disregard the others.
6. Then analyze the droplet in the picture to get the info for integrated density, mean gray value, and area.
7. Repeat steps for one picture of each sample.
8. Save your data in an excel page for easy access at another time
Research and Development
Specific Cancer Marker Detection - The Underlying Technology
- Our genes can tell us anything and everything about ourselves.
- The sooner we can detect cancer, the more effectively it can be prevented or treated.
- So why not find out about our disposition to cancer with our genes?
- The science community has identified many DNA sequences that are correlated to incidence of cancer. Through a process known as Polymerase Chain Reaction, (or PCR,) we can make tons of copies of any sequence of DNA from a DNA template. So, let's say we want to find out if someone has a sequence of DNA that may be indicative of a higher cancer risk; how can we do it?
r17879961 is a sequence of DNA that has been positively linked with cancer. It is a part of a sequence of DNA that codes for a protein kinase called CHEK2.
Bayes' Law Pr(A|B) = Pr(B|A)*Pr(A) / Pr(b)
Pr is the probability, where A is an event and B is another event. The equation calculates the probability of an event happening if another event is true.
This image depicts the amplification of a DNA sequence. (An original picture made by our group on PowerPoint)
ImageJ Software Processing
| Sample || Integrated Density || DNA μg/mL || Conclusion
| PCR: Negative Control || 1658372 || 0 || Negative
| PCR: Positive Control || 3527419 || 2 || Positive
| PCR: Patient 1 ID 19185, rep 1 || 1173985 || 0.82 || Negative
| PCR: Patient 1 ID 19185, rep 2 || 1600503 || 1.02 || Negative
| PCR: Patient 1 ID 19185, rep 3 || 1621751 || 1.21 || Negative
| PCR: Patient 2 ID 88142, rep 1 || 927301 || 0.20 || Negative
| PCR: Patient 2 ID 88142, rep 2 || 962092 || 0.23 || Negative
| PCR: Patient 2 ID 88142, rep 3 || 901293 || 0.19 || Negative
- Sample = Comprised of two drops of DNA solution and two drops of SYBR Green.
- Integrated Density = The integrated density of the drop minus the integrated density of the background. This calculation account for background noise.
- DNA μg/mL = The integrated density of sample divided by the integrated density of drop multiplied by two.
- Conclusion = If the reading is positive then it means that the cancerous mutation is present.If the reading is negative or "no signal," then the mutation is not present.