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Lab Write-Up 1
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Lab Write-Up 3
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LAB 1 WRITE-UP
Initial Machine Testing
The Original Design
When the LCD screen was unplugged from the Open PCR brains Board, the information for the sample cycle was no longer available on the screen.
When we unplugged the white wire that connects the brain board to the 16 tube PCR block, the machine temperature was unable to be regulated.
The initial test date of the Open PCR Machine was October 24, 2012. Our experience with the machine was rather time consuming, with the experiment taking an hour and a half to complete. However, the machine was very straightforward and easy to use.
Polymerase Chain Reaction
The Polymerase Chain Reaction is a process controlled by thermal cycling; a system of repeatedly heating and cooling the sample. It is simple and inexpensive, as PCR allows you to make millions of copies of DNA to better analyze data. First you need a small amount of DNA and a small amount of some PCR reaction mix. A program on the machine needs to be created consisting of three stages. During the three stages, the DNA is first heated to separate the DNA double helix, creating two single stranded DNA molecules. The thermal cycler is now cooled and primers match up to the DNA strands before they naturally attempt to pair up. As the cycles progress, DNA is replicated and used as a template to create additional copies.
There are several components of a PCR reaction. The template DNA amplifies millions of copies to determine if they are cancerous. The many primers start the binding of complentary strands (specific primers) and they bind to cancer sequences. The taq polymerase is a protein that catalyzes the DNA assembly. There is also a cofactor, which binds to Taq to enable optimal binding speed. Lastly, there are deoxynucleotide tri-phosphates, which builds a new strand of DNA.
Steps to Describe How to Amplify a Patient's DNA Sample:
Step 2: Heat Denaturation
Step 3: Primer Annealing
Step 4: Primer Extension
Step 5: Termination
NOTE: 8 samples total: 3 trials (a, b, & c) of each patient (1 & 2) with a positive control and a cancer DNA template.
1. Empty the given container of the separate parts for the fluorimeter.
2. To create a darker environment for the images, unsnap one side of the container and place the container upside-down so that the unsnapped flap of the container flips up.
3. Place the smartphone to be used for the experiment into the given black stand to provide a stable stand for the camera.
4. Position LED box with the drop in the back of the container with the camera parallel to it so that the camera is viewing light passing through the water droplet.
1. Inactivate the designated camera's flash
2. Set ISO to 800+
3. Set white balance to auto
4. Set to max exposure
5. Lower saturation to a minimum
Research and Development
Specific Cancer Marker Detection - The Underlying Technology
(See above for PCR explanation)
CHEK 2, code rs17879961, is a single nucleotide polymorphism (SNP) that is linked to cancer. It is a symbol for CHK2 Checkpoint Homolog and is located on chromosome 22. Mutations of the CHEK2 gene are related to increased risk of breast cancer. In week three's class, it was concluded that PCR can be used to bind specific DNA primers to the cancerous DNA bases--resulting in cancerous DNA amplification. As a result, the PCR reaction will form normal DNA sequences and the supposed cancerous CHEK2 strain. Possible primers for supposed amplification were used in the experiment and were compared to sample sequences to determine if the DNA in question was amplified.
Extra information: Gene missense, T mutates to cancer associated C for the specific allele. Cancer sequence binding primer: AACTCTTACACTGCATACAT. In this scenario, the risk allele in the highlighted C base.
Where C is the cancer present and T is the positive test. Other values include the following: P(C) is prior probability and P(T|C) and P(T|~C) are conditional probabilities.
Bayesian reasoning is a conditional probability tool used to help understand the limitations of diagnostic testing. The formula for Baye's Rule is given above--it is also given in various forms. Through Baye's reasoning, four important things can be calculated: Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV). Sensitivity is the probability that an individual with the disease will result in a positive, and specificity is the probability that he/she without the disease will result in a negative test. PPV refers to the probability that an individual with a positive test actually has the disease, where as NPV is the probability that an individual with a positive test does not actually have the disease. It is important to utilize Baye's Rule because it will give a better understanding to the reliability of open PCR results based on the probabilities of false positives and false negatives.