LAB 1 WRITE-UP
Initial Machine Testing
The Original Design
Displayed above, the OpenPCR Machine is used to amplify DNA in a short amount of time. PCR, or polymerase chain reaction, is a process that essentially replicates a small amount of DNA until there is a substantial amount present for testing. The DNA in the machine is subject to dynamic environment changes, as the machine rapidly heats and cools the single stranded sample until multiple copies of the original DNA are created. The OpenPCR Machine is connected to a computer via a USB cable, which allows the replication process to be controlled entirely on the computer screen. In order to run an experiment, temperatures and a desired amount of cycles must be entered on the computer, info which is then transmitted to the machine in order to run the process. After the inputted information is received by the OpenPCR Machine, the DNA inside will be subject to the exact amount of cycles and temperature conditions that were set on the computer. The machine will then read results of the completed process which are clearly displayed on an LCD screen on the OpenPCR mechanism itself.
Experimenting With the Connections
When we unplugged (part 3) from (part 6), the machine failed to display any information on the LCD projector. This is evidence that part 3 and part 6 connect the circuit board to the LCD display, so when unplugged, the machine was unable to display any results.
When we unplugged the white wire that connects (part 6) to (part 2), the machine displayed a temperature that was lower than the actual temperature inside the system. This is evidence that part 2 and part 6 connect the circuit board to the temperature pad, so when unplugged, the PCR machine failed to display accurate temperature readings.
The first test run of OpenPCR Machine 1 was conducted at 10:00 a.m. on October 23, 2013. The machine ran silently for two hours, and no complications were present. Data displayed on the LCD screen corresponded with data displayed on the computer software program, and the data displayed a steady increase in temperature as time went by. This is because the DNA needed to be raised to a temperature of 95 degrees Celsius to begin the process of copying. However, even though our machine displayed data that was consistent on both the computer and the machine itself, our team realized that a substantial amount of time is needed to run multiple cycles and produce enough duplicates of the targeted section, as each cycle takes around three to four minutes to complete.
Thermal Cycler Program
DNA Sample Set-up
| Positive Control: |
Tube Label: PC
| Patient 1 |
Tube Label: 1A
| Patient 1 |
Tube Label: 1B
| Patient 1 |
Tube Label: 1C
| Negative Control: |
Tube Label: NC
| Patient 2 |
Tube Label: 2A
| Patient 2 |
Tube Label: 2B
| Patient 2 |
Tube Label: 2C
DNA Sample Set-up Procedure
1. Gather the sixteen mixes.
2. On ice, prepare eight test tubes of the reaction mix on ice. 25ul PCR reaction mix, 25ul primer mix.
3. Place the eight test tubes into the PCR machine.
4. Let the machine run its indicated number of cycles. Record results from the L.E.D. screen, and remove the eight test tubes from the machine at the conclusion of the experiment.
PCR Reaction Mix:
The PCR reaction mix contains bacterially derived Taq DNA polymerase, MgCl2, dNTP's, and reaction buffers.
DNA/ Primer Mix:
Each of the DNA/primer mixes contains a different template DNA. All of the tubes have the same forward primer and reverse primer.
Research and Development
PCR - The Underlying Technology
Component Function of a PCR Reaction
A PCR reaction is composed of multiple components, each serving its own unique purpose. The building block of the reaction, the template DNA, is a sample that is replicated into multiple copies. Once replicated, there will appear to be many identical strands of the template DNA. The component that synthesizes the copies of template DNA is called the taq polymerase. Taq polymerase is an enzyme that is thermostable, so it won't denature when temperatures rise (as they do in PCR reactions). The polymerase begins synthesizing DNA strand once it receives signal from the primers, another component of a PCR reaction. The reason the primers are essential to the reaction is because the polymerase won't begin without them. The primers also make the sequence more unique, allowing the polymerase to only synthesize according to the specific sequence placed by primer. Also important in a PCR reaction is magnesium chloride (MgCl2). The MgCl2 severs as a co-factor to the taq polymerase as some enzyme cannot function without certain co-factors. The last important component in a PCR reaction is deoxyribonucleotides (dNTP's). These dNTP's compliment the template DNA and control mis-priming. Without correctly matched dNTP's to the template DNA, a new strand cannot be formed.
Steps of Thermal Cycling
The majority of PCR reactions use a process called thermal cycling in which the reaction is cyclicly heated and cooled to allow the DNA to melt and the enzymes to replicate. In these thermal cyclers, a heated plate is inserted above the test tubes to prevent condensation at the top of the test tubes. During the initial step of the cycle, the DNA is heated to 95°C degrees Celsius for three minutes, during which the DNA is unwound and placed in a parallel line. The DNA is then held at 95°C for another 30 seconds so that the DNA strand is broken into two separate pieces, which is known as the denature stage. During the anneal step, the DNA is cooled to 57°C for 30 seconds to allow the primers to attach to the strands, preparing for the polymerase to begin synthesizing. The DNA is then heated again into the extend step, in which the taq polymerase binds to the template DNA and begin to replicate. This step occurs for 30 seconds at a temperature of 72°C. Finally, the test tubes are kept at 72°C for three minutes to allow the taq polymerase to identify and find all of the primers in the DNA so that it ensures that it synthesized all the DNA it needed to. Once the final three minutes has passed, the tubes are held at 4°C to detect the amount of DNA present in the sample.
There are four different bases, or nucleotides, that compose a DNA strand. The four bases are as follows: Adenine (A), Thymine (T), Cytosine (C), and Guanine (G). These nucleotides are bonded to their complimentary base on the template DNA strand by hydrogen bonding to create a double-helix DNA strand. The nucleotides can only attach to a different specified nucleotide; adenine bonds to thymine (and vice versa), and cytosine bonds to guanine (and vice versa).
Primers on Cancer DNA