BME103:T930 Group 2

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

OUR AWESOME TEAM

Ryan SullivanResearch Development Scientist
Ryan Sullivan
Research Development Scientist
Miriam AcostaPCR Machine Engineer
Miriam Acosta
PCR Machine Engineer
Ryan KeeneyPCR Machine Engineer
Ryan Keeney
PCR Machine Engineer
Juliana RamosExperimental Protocol Planner
Juliana Ramos
Experimental Protocol Planner
Aaron CornejoExperimental Protocol Planner
Aaron Cornejo
Experimental Protocol Planner

LAB 1 WRITE-UP

Initial Machine Testing

Open PCR.
Open PCR.


The Original Design
An Open PCR machine is a thermo-cycler used to cycle DNA. By rapidly and precisely changing the temperature through a heating plate and cooling fan, the machine manages to oscillate the temperatures of the DNA samples. The low-cost machine runs through software that is user-friendly and initiates the process once the heated lid is properly closed on top of the DNA samples. The samples are placed in the designated sample holders and the computer program commences the process. The PCR goes through a cycle of heat changes for a specified duration of time in order to facilitate the replication and amplification of DNA sample.


Experimenting With the Connections

Unplugging the LED interface cable from the processing chip caused the display of the machine to turn off. The blank screen meant that the machine stopped sending data to display. Unplugging anything from the circuit board causes it not to work because no electricity can be sent.
When the white wire was disconnected from the circuit board, the PCR machine discontinued to record temperatures correctly. Thus making the Open PCR machine impractical for its actual function.


Test Run

Our initial testing of the PCR machine went quite well. We used the Open PCR software to program our custom cycle quite easily. Once the PCR was connected to the computer via USB we continued to run the trial. During the run we made sure that the temperature displayed on the LED display match the temperature on the computer program. The temperatures matched the entire duration of the experiment. The test run carried out flawlessly, just a bit slowly.




Protocols

Polymerase Chain Reaction
A PCR (polymerase chain reaction), according to Cold Spring Harbor Laboratory, “enables researchers to produce millions of copies of a specific DNA sequence in approximately two hours. This automated process bypasses the need to use bacteria for amplifying DNA.” Thus forth, it essentially does go through a biochemical process to replicate the desired sequence of DNA. The PCR machine has the capacity to isolate and amplify that desired strand of DNA through the repeated cycling of cooling and heating.


Thermal Cycler Program:

Stage one: 1 cycle, 95°C for 3 minutes

Stage two: 35 cycles, 95°C for 30 seconds, 57°C for 30 seconds, 72°C for 30 seconds

Stage three: 72°C for 3 minutes

Final hold: 4°C


Reagant 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
dH2O 47.8 μL
Total Volume 100.0 μL


Patient 1
Sample ID Sex Age Date/Time
Control 15308 M 61 Thursday 9:30 AM
1-1 15308 M 61 Thursday 9:30 AM
1-2 15308 M 61 Thursday 9:30 AM
1-3 15308 M 61 Thursday 9:30 AM
Patient 2
Sample ID Sex Age Date/Time
Control 36372 F 56 Thursday 9:30 AM
2-1 36372 F 56 Thursday 9:30 AM
2-2 36372 F 56 Thursday 9:30 AM
2-3 36372 F 56 Thursday 9:30 AM


Flourimeter Measurements

Droplet of water on hydrophobic slide.
Droplet of water on hydrophobic slide.
Proper set up.
Proper set up.

Flourimeter Assembly Instructions:

1. Gather materials (PCR Machine, tools optional)
2. Remove the lid from black box and flip-flop the box
3. Open the front flap of the black box.
4. Place hydrophobic/glass slide
5. Using pipette to obtain droplets of SYBR green dye
6. Proceed to squeeze two drops of dye on the slide.
7. Obtain DNA sample.
8. Add DNA sample to the SYBR green dye.
9. Set up glass and box as shown in the picture.
10. Make sure the blue LED light shines through the droplet.
11. Take picture with smartphone (should be set up as indicated in step 9)
12. Double check thatthe stand and the flourimeter is covered by the box to make sure not light comes in.
13. Take a well focused photo with smartphone.
14. Finally, use ImageJ to obtain information from the picture.





Research and Development

Specific Cancer Marker Detection - The Underlying Technology

The r17879961 DNA was sequenced to determine the chain of nucleotides that it is composed of. A section of this DNA shows a change in the allele sequence; one which might indicate cancer. In the sequence, a change from "ATT" to "ACT" is the specific indication marker for cancer. The artificial primer that is introduced to the DNA chain is meant to be a complimentary strand that matches up with the "ACT" and surrounding nucleotide sequence ("TGA"). When the primer attaches successfully, it allows the Taq polymerase to finish attaching nucleotides to the sequence. The number of strands will be doubled in this case, and by using the PCR replication machine, the sample will be heated and cooled in specific intervals. These intervals are meant to manipulate the DNA and do things such as separate a strand, cool it down so it is able to bind, as well as stretch it out. This process will, ultimately, result in exponential DNA replication. If the primer does not attach successfully, then this means the person does not have a cancer-indicating sequence, and it will, therefore, not replicate the DNA exponentially. If the DNA replicates exponentially, then the fluorescent dye will show, and this would indicate a cancerous genome.


After the PCR machine replication, the samples were put on a slide and combined with Sybr Green indicator. They were then placed in a dark box and a picture was taken of each sample. The samples pictures were uploaded onto the computer and we were able to use software to measured the amount of green fluorescent dye that was in the replicated DNA. The data in the section below describes the data and the parameters that indicate cancer using this information.


The illustration below shows the PCR process when the primer attaches to the DNA and the Taq polymerase and is able to complete the DNA chain, in coordination with the PCR machine temperature cycles. It is shown that throughout the process, the DNA replicates at an exponential rate.


*Imagine from above with labels


With the r17879961 DNA that was sequenced, the primer that was made for the DNA contained a strand of nucleotides that went: "ATGTGACGT".

These matched up with the section of the DNA in the 22nd chromosome if the sequence did indicate cancer. The cancer sequence would be complimentary to the primer, it is: "TACACTGCA". This would yield a positive result

A non-cancerous sequence is shown below ("TACATTGCA"), which would not attach to the primer and would yield a negative result.

When the primer attaches to the cancerous sequence, it works, and the replication process can begin:

When the primer tries to attach to a non-cancerous sequence, it fails, because the "T" and "G" do NOT connect, and therefore the replication process is not exponential:



Results

Positive Control
Positive Control
Negative Control
Negative Control



Patient ID 15308 Sample 1
Patient ID 15308 Sample 1
Patient ID 15308 Sample 2
Patient ID 15308 Sample 2
Patient ID 15308 Sample 3
Patient ID 15308 Sample 3


Patient ID 36372 Sample 1
Patient ID 36372 Sample 1
Patient ID 36372 Sample 2
Patient ID 36372 Sample 2
Patient ID 36372 Sample 3
Patient ID 36372 Sample 3



Sample Integrated Density DNA μg/mL Conclusion
PCR: Negative Control 1337080 0.521457349 No Signal
PCR: Positive Control 3951896 1.541228058 Positive
PCR: Patient 1 ID 15308, rep 1 4456673 1.738089634 Positive
PCR: Patient 1 ID 15308, rep 2 3545161 1.382602579 Positive
PCR: Patient 1 ID 15308, rep 3 3228305 1.259029652 Positive
PCR: Patient 2 ID 36372, rep 1 960771 0.374697923 No Signal
PCR: Patient 2 ID 36372, rep 2 898943 0.350585181 No Signal
PCR: Patient 2 ID 36372, rep 3 742539 0.289588071 No Signal


KEY

  • Sample = Describes sample number and patient
  • Integrated Density = The Integrated Density of the Drop minus that of the background
  • DNA μg/mL = 2 * IntDen of Sample/IntDen of Calf Thymus (both with background subtracted out)
  • Conclusion = Whether exponential DNA replication has occured

Given the results of all the DNA testing and evaluation we conclude that Patient 1 (ID 15308) contains the cancerous gene tested for, and that Patient 2 (ID 36372) did not.



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