BME103:T130 Group 17

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GROUP17

Jorge Jimenez Research Development Specialist
Jorge Jimenez
Research Development Specialist
Ricardo Robles Research Development Specialist
Ricardo Robles
Research Development Specialist
Jennifer Begin Experimental Protocol Planner
Jennifer Begin
Experimental Protocol Planner
Finola Roy Experimental Protocol Planner
Finola Roy
Experimental Protocol Planner
Kevin Zenner Open PCR Machine Engineer
Kevin Zenner
Open PCR Machine Engineer

LAB 1 WRITE-UP

Initial Machine Testing

The Original Design
(Add image of the full OpenPCR machine here, from the Week 3 exercise. Write a paragraph description for visitors who have no idea what this is)
Image of the Open PCR

Experimenting With the Connections

When the PCB board of the LCD screen was disconnected from the PCB circuit board the display output was turned off.

When the white wire connecting the 16 tube PCR block to the PCB circuit board ability to regulate the temperature of the PCR was lost.


Test Run

October 25, 2012

After finishing the diagnostic analysis, the PCR was tested by setting thermal cycler program to three stages. Stage one was one cycle of 95 °C for 3 minutes, the second stage was 35 cycles, 95 °C for 30 seconds, 50 °C for 30 seconds, 72 °C for 30 seconds, stage three was one cycle of 72 °C for 3 minutes. The test run lasted for about an hour and thirty minutes and confirmed that the temperature readings on the LED of the PCR machine and the computer matched.




Protocols

Polymerase Chain Reaction

The PCR replicated the wanted DNA fragments from the patient. The PCR will heat up to 95 degree Celsius and cool down to 50°C. Then heat back up to 72°C within one cycle. Over all there will be 30 cycles. At the end of the 30 cycles we have over a billion of the wanted fragments, and 60 unwanted DNA molecule strands in the solution.

Step by Step instruction to amplify the Patient's DNA Sample:

1. Need to extract the DNA from the patient.

2. Put the DNA into a special PCR tube.

3. Add primer #1 to the PCR tube with the DNA.

4. Add primer #2 to the PCR tube with DNA.

5. Add Nucleotides (the A,C,T,and G).

6. Add the DNA polymerase to the PCR tube.

7. Place the PCR tube into the thermal cycler.

8. Set the temperature of the thermal cycler to 95°C and set the machine to run 30 cycles.

9. Now the thermal cycler cools down to 50°C and primer #1 and #2 attach to the single strands of DNA.

10. Now the thermal cycler temperature changes to 72°C. This begins the DNA polymerase. This pairs the DNA with its complimentary nucleotide through to the end of the DNA strand.

11. Repeat step 8-10 29 more times.

12. During cycle #3 the wanted DNA begins to appear.

13. The wanted piece of the DNA begins to double.

14. After 30 cycles are complete over a billion wanted DNA fragments will show in the DNA solution and there will be 60 copies of unwanted DNA molecules in the solution.

Components:

1. MgCl_2

2. Taq DNA polymerase that lacks 5'--> 3'

3. dNTPs

4. Reaction buffers

Reagent and Volume Table

Reagent Volume
Template DNA (20 ng) 0.2 μL
10 µM forward primer 1.0 μL
10 µM reverse primer 1.0 μL
GoTaq Master Mix 50.0 μL
dH_2O 47.8 μL
Total Volume 100.0 μL

Description of samples:

Image 1: 3 drops of sybrgreen, 2 drops of calibrator solution. Dot was all blue not green. This implies the solution is negative for cancer.

Image 2: 4 drops of sybrgreen, 2 drops of water solution. Dot was all blue no green. This implies the solution is negative for cancer.

Image 3: 3 drops of sybrgreen, 2 drops of patient 1 solution A. Dot has some slight blurrs of gree. This implies the DNA solution is positive for cancer.

Image 4: 2 drops of sybrgreen, 2 drops patient 1 solution B. Dot was all blue with no sight of green. This implies the DNA solution is negative for cancer.

Image 5: 3 drops of sybrgreen, 2 drops of patient 1 solution C. Dot was all blue with no sight of green. This implies the DNA solution is negative for cancer.

Image 6: 3 drops of sybrgreen, 2 drops of patient 1 solution D. Dot was all blue with no sight of green. This implies the DNA solution is negative for cancer.

Image 7: 3 drops of sybrgreen, 2 drops of patient 2 solution A2. Dot was all blue with no sight of green. This implies the DNA solution is negative for cancer.

Image 8: 3 drops of sybrgreen, 2 drops of patient 2 solution B2. Dot has a spec of bright green. This implies the DNA solution is positive for cancer.

Image 9: 4 drops of sybrgreen, 2 drops of patient 2 solution C2. Dot had some spattered green. This implies the DNA solution is positive for cancer.

Image 10: 5 drops of sybrgreen, 2 drops of patient 2 solution D2. Dot had a really green center. This implies the DNA solution is positive for cancer.

Patient ID:

Patient 1: 74065, Male, Age:63

Patient 2: 64835, Male, Age:46


Flourimeter Measurements

1. Turn on the blue light in the Flourimeter using the switch for the Blue LED.

2. Place the smart phone accordingly so that the super-hydrophobic slide is in front of the smart phone.

3. Turn on the camera on the smart phone. **TURN OFF THE FLASH** Set the ISO to 800 or higher. Increase the exposure to maximum.

4. Set the distance between the smart phone and the machine so that the smart phone can take a clear picture of the droplet.

5. First label the blank pipettes according to the patients (A,B,C,D... all eight of them). The pipettes given by the instructor are color coded. The white coded pipette is used for water. the red coded pipette is used for the calibrator (the tube with the red dot). The blue coded pipette is used for the sybrgreen (the tube with the blue dot). The black coded pipette is used to pick up the waste and put it in the cup that collects the waste droplets.

6. First calibrate the machine (to make sure the machine works). Put two droplets of the cyber green on the first two dots in the middle. If the droplets are not connected then add a third droplet that combines the two droplets.

7. Then put two drops of calibrator solution with the calibrator solution. Then set the smart phone accordingly to take a clear picture of the droplet. Then put the black box on top of the phone and the machine so that when the the light is completely blocked and the shade of blue of green is shown when the picture is taken.

8. Record observations.

9. Remove the solution from the glass dish using the black pipette and discard it in the plastic cup given for the waste droplets.

10.Repeat step 6-9, but instead of adding calibrator solution, add 2 droplets of water.

11.Repeat steps 6-9 for patient 1 solutions (A,B,C,D) and patient 2 solutions (A2.B2,C2,D2).



Research and Development

Specific Cancer Marker Detection - The Underlying Technology

The r17879961 sequence will produce a cancer mutation at Chromosomes 22 of the gene sequence. The normal sequence has a T ( thymine) nucleotide at chromosome 22 while the mutation sequence has an associated C (cytosine) nucleotide. The Open PCR machine is able to determine whether or not the r17879961 sample has cancer by replicating the desired mutation exponentially. Positive and negative strands are inserted into the PCR with a certain primer. The primer in the reaction is designed to attach to the C nucleotide that signifies cancer mutation. One strand has the primer, while the other strand does not. Open PCR will replicate the strand with the certain primer, causing an exponential growth. The negative strand will grow in a linear fashion. The PCR process goes through 30 cycles to complete this. After the PCR process, fluorescent dye is added to the solutions. The fluorescent dye will cause the DNA with double strands to glow. Since the PCR has grown the double stranded positive DNA exponentially the fluorescent dye glows brighter. Therefore the cancer DNA is in the sample with the glow.


First Step The DNA double helix seperates, creating two single-stranded DNA molecules.
Second Step Primers attach and lock onto the targeted sequence before the strands can rejoin.
Third Step DNA polmerase locates the primerr and begins to add complimentary nucleotides onto the strands.

Fourth Step Cycle one is complete.

Fifth Step The steps are repeated, increasing the amount of DNA.

Sixth Step Primers attach to the targeted sequence

Seventh Step The desired fragments are segments of the targeted DNA (Cancer).

Eighth Step These fragments will continue to duplicate with each cycle.

Source: Genetic Science Learning Center (2012, August 6) PCR Virtual Lab. Learn.Genetics. Retrieved November 8, 2012, from http://learn.genetics.utah.edu/content/labs/pcr/

Results

Sample Integrated Density DNA μg/mL Conclusion
PCR: Negative Control 2003444 0 negative
PCR: Positive Control 9972359 2 positive
PCR: Patient 1 ID 74065, rep 1 7454123 1.495 positive
PCR: Patient 1 ID 74065, rep 2 5846771 1.1726 positive
PCR: Patient 1 ID 74065, rep 3 3886745 0.7795 negative
PCR: Patient 2 ID 64835, rep 1 26052159 5.2249 positive
PCR: Patient 2 ID 64835, rep 2 33652771 6.7492 positive
PCR: Patient 2 ID 64835, rep 3 48574317 9.7418 positive


KEY

  • Sample =
  • Integrated Density =
  • DNA μg/mL =
  • Conclusion =


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