Our re-design is based upon the Open PCR system originally designed by Josh Perfetto and Tito Jankowski.
System Design
Key Features
As one can see in these two images, the picture features the PCR machine, stripped of everything internal except for the exhaust fan. Our idea of improving this machine is to add an additional fan, to deal with the buildup of heat. The PCR machine is designed to heat up the samples, increasing internal temperature due to the trapped heat. While the exhaust fan does assist in keeping a reasonable temperature, the hot air it removes is only directly being pushed out, there is no active air flow through the machine, which would cool it better, only an active air flow out of the machine.
While only pushing air out works, adding an extra fan opposite of the exhaust fan would create air flow through the entire machine, pulling in room temperature air, allowing better circulation. If the fan were added on, there is no reason that the machine could not keep a constant temperature only a couple degrees above room temperature.
Instructions
Protocols
Polymerase Chain Reaction
Polymerase chain reaction is basically molecular photocopying and the process or technique used to make copies of small segments of DNA because it only targets specific segments of the DNA and that's what makes it useful. PCR works by mixing two DNA fragments, also known as primers which are about 20 bases long. The mixture is then heated and denatured and then the primers bind to their complementary sequences on the separated strands. Then, the polymerase extends primers into new complementary strands and it goes through about 30 cycles. PCR products are useful and can be used in many experiments like DNA fingerprinting and detection of viruses.
Collect three replicate DNA samples from two patients. (was provided already)
The DNA samples are 50 μL each, get the patient's ID and label the the each tube.
PCR reaction mix - Mix contains Taq DNA polymerase, MgCl2, dNTP's, forward primer, and reverse primer.
The primers are artificial DNA, designed to match the chain of DNA we want.
Taq polymerase is the enzyme that binds to the end of the new chain and recreates the separated DNA.
Mgcl2 binds to Taq as a co-factor and helps Taq to function appropriately, and affects the speed of the Taq binding to the loose strands.
dNTp's is dioxnucleotidetriphosphate. this is what is used to recreate the second DNA strands.
The 8 tubes of mixtures will then go through the cycles in the PCR system.
During each step of the thermal cycling, the DNA is unzipped and heated to 95°C to break the H-bonds between the 2 strands. This exposes the part we want in this lab experiment. Then, the primer binds to the trage we want without cancer marker, this primer won't bind. Next, the temperature will be dropped to 57°C in order to bind the primer. Later, it is heat it back up to 72°C with the Taq to reform and duplicate DNA strands. Finally, this thermal cycling is repeated for amplification and add dye that binds specifically to DNA for detection.
Pippette 50 μL DNA sample into the labeled PCR tubes.
Place the pipettes separately on the table to avoid contamination
Use a pipette to add 50 μL of GoTaq Master mix to each of the PCR tubes, and discard the pipette after each tube.
Create a new program on the Open PCR system (connected to the computer)
Create three stages
Stage 1: 1 cycle, 95°C for 3 minutes
Stage 2: 30 cycles for 95°C for 30 seconds, 57°C for 30 seconds, 72°C for 30 seconds
Stage 3. 72°C for 3 minutes
Final hold: 4°C
Save the Program
Place PCR tubes inside the machine and push down on the lid to close it
Finally, click the start button in the OpenPCR program and run the experiment.
The time for this experiment will be reduced due to the improvements made on the PCR machine. It should cycle much quicker than it did before (about an hour and a half to two hours). How much of a time difference is uncertain.
The Components of the GoTaq® Colorless Master Mix
"dNTP's, MgCl2, and reaction buffers at optimal concentration for efficient amplification of DN templates by PCR."
Volumes Used for Mixture
Table 1
Reagent
Volume
Template DNA (20 ng)
10.2 μL
10 μM reverse primer
1.0 μL
dH2O
47.8 μL
0 μM forward primer
1.0 μL
GoTaq master mix
50.0 μL
Total Volume
100.0 μL
DNA Samples (8)
Positive Control: Cancer DNA Template Tube label A
First, the glass side of the slide was placed faced down onto the device.
A different pipette was used for transferring each content from the small tubes to the bigger ones.
After labeling the tubes and pipettes, gloves were worn to ensure a contamination free procedure.
Using the specific pipette for each component, one drop of buffer was put onto the first and second centered holes of the slide and two drops of diluted sample were placed on the gathered buffer drops.
Turn on the light and adjust it so that it goes through the drop.
The device was then put as far back inside the upside down box as possible so that much of the stray light will be removed.
The phone was placed into the holder inside the box.
After customizing the photo settings in the phone according to the instruction sheet, a shot of the drop sample was taken and saved.
The number of the photo was recorded in a table to keep track of the photos.
The photo was sent to the e-mail of the group member who was responsible for analyzing the photo.
The previous steps were repeated for each sample with the exception of:
Replacing the water drops with the rest of the samples.
Using next two consecutive holes on the glass slide each time a sample was used.
Open ImageJ
1. By using a USB cable, connect the camera phone to the desired computer that has already ImageJ installed.
2. Under my computer, choose portable devices where you could find the smartphone listed; double-click on it.
3. After localizing the DCIM folder and opening it, you should select camera.
4. The desired photos can then be transferred by simply putting them into the created folder.
5. Open ImageJ and go to file; click on it and choose open.
6. Select browse then pick the desired picture from the same folder created earlier.
7. To continue opening different pictures, you should only repeat steps 5 and 6.
Research and Development
Background on Disease Markers
Parkinson’s Disease is a degenerative disorder in the nervous system where the nerve cells cannot send messages to the muscles adequately due to a lack of dopamine. This usually leads to tremors and a difficulty moving. Typically, Parkinson’s disease develops in person after the age of 50, but it is not always the case. This disease cannot be cured, but it can be treated.
This disease is generally contracted through genetics. The SNP of this is can be found in SNP cluster report of rs2853826. The error is due to an adenine nucleotide replaced with the guanine.
Nehal, here is your part that you need to rewrite in your own words and apply to Parkinson's disease. Also, an A has been replaced by a G in this case.
"In this particular instance, it is a adenine replaced by the cancerous-related nucleotide cytosine as shown above as the bold C. The primer will only bind to the matching sequence (testing sequence) because of the A-T, and C-G pairings. Since an A has been replaced with a C, the primer can't bind to the DNA strand beyond that specific nucleotide in the sequence. Due to the open double helix, further steps in the reaction will not happen, and no amplification will happen. No amplification means no visible results and the test will run negative.
If the cancer gene is present, then the matching primer will completely bind to the DNA strand. When this happens, the amplification sequence will be able to precede, and this will show up as a positive result."
Image by: Alyssa Alexander
This is all in theory of course, and should work perfectly every time. But there are many factors to consider. For instance, not every one who has cancer has the cancer gene.
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BME 103 Fall 2012
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