BME103 s2013:T900 Group1
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Lab Write-Up 1
Lab Write-Up 2
Lab Write-Up 3
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LAB 1 WRITE-UP
Initial Machine Testing
The device displayed above, commonly referred to as an "Open PCR machine," is a mechanism that performs polymerase chain reactions that amplify DNA by creating a dynamic environment with extreme temperature changes. Upon undergoing mutilpe cycles of continual heating and cooling, the DNA present within the samples are separated then rapidly replicated resulting in multiple copies from a single strand of DNA. The PCR machine is hooked up to a computer using a usb cable allowing the user to control the desired number of cycles and temperatures for an experiment. Once the information is submitted into the system, the DNA experiences environmental changes controlled by the heating plate, heating lib, and cooling fan. The circuit board towards he bottom of the device then computes the information and transfers it to the LED screen presenting immediate results.
When we unplugged the LCD screen(part 3) from circuit board (part 6), the machine didn't display any information on the LCD screen.
When we unplugged the white wire that connects Circuit board (part 6) to heat block (part 2), the machine didn't read the temperatures accurately.
(Write the date you first tested Open PCR and your experience(s) with the machine)
PCR, or Polymerase Chain Reaction, is a method of amplifying a particular segment of DNA by way of selective replication. If the sequence of the target DNA segment is known, primers can be created from the complimentary base pairs at each end of the target segment. A forward as well as a reverse primer is required so that each double strand of DNA results in two double strands of DNA at the end of each cycle (as Taq DNA Polymerase only works in one direction, it needs a primer at the beginning of both strands in order to copy both). To complete PCR, a DNA sample (containing the target DNA segment) must be mixed with the primers (both forward and reverse as discussed above), Taq DNA Polymerase, dNTP's (free base pairs to be used as the building blocks in the new DNA put together by the polymerase), and MgCl2 (a required substrate for the polymerase to use the dNTP's).
DNA Sample Set-up
DNA Sample Set-up Procedure
PCR Reaction Mix
DNA Sample/Primer Mix
Research and Development
Specific Cancer Marker Detection - The Underlying Technology
Polymerase chain reaction (abbreviated PCR) is the process by which a selected strand or segment of DNA is replicated various times. Each step in the process is vital for the process to occur efficiently.
In the first step of PCR where the samples were heated to 95ºC for three minutes, the two strands of DNA were separated from one another due to the high amount of heat in the reaction. This temperature and the amount of time that the samples were heated were both necessary because they allowed for an adequate amount of energy necessary to break the hydrogen bonds between the parallel nucleotides. This caused the DNA sequence to unzip, which opens up the opportunity for the entire reaction to occur. After this occurred, 35 cycles of the samples being heated up to 95ºC, down to 57ºC, down to 72ºC occured. Again, at 95ºC the DNA was separated in order for the individual components to begin mixing together, like a soup. At 57ºC, the samples were cooled down, which allows for the forward and reverse primers to bind to their respective regions. When reheated to 72ºC, the TAQ polymerase was activated which generated a new strand of DNA from both the lagging and leading strands. The primers tell the polymerase where to begin generating new sequences of DNA, with the Magnesium Chloride acting as a catalyst for this reaction. At 72ºC, the temperature is adequate enough to where the polymerase can generate a new strand of DNA from the template quickly without denaturing. After this final step, the new sequences of DNA are fully generated and the process begins over again 35 times, each resulting in two new DNA strands.
This helps us because only the cancerous DNA is replicated, whereas the original template DNA is not replicated at all. This is because the primers are designed to bind only to the cancerous sequences of DNA. This is because that specific type of cancer's DNA sequence was analyzed and a primer generated that binds to that analyzed, cancerous sequence. As such, when the primers detect this, they bind to those, and the DNA polymerase begins generated a sequence of DNA for this strand. This does not occur in the normal strand of DNA, simply because it doesn't have that specific sequence, and as such the primers won't bind to it. The mutation in the cancer allows for those specific primers to bind to that sequence, but the opposing, normal strand will not bind to it, even when the strand is continuously copied because the mutation isn't present there. As such, the cancerous sequence will be replicated, while the normal strand remains uncopied.
This image shows exactly how the process works. In the beginning, at the top left, is the template DNA once it starts up. The second image down shows what occurs when the temperature is increased, then decreased. The template opens up allowing for each component of the PCR to go between the two strands, and once it's cooled down, the primers bind to each corresponding end of the DNA sequence that matches it. Keep in mind, that these primers were designed to bind only to the segments that have the corresponding cancer gene, otherwise it won't bind to them. What occurs next is when the temperature goes up, which is the next two images below. TAQ polymerase is activated, goes to the primer, and begins creating a segment of new DNA using the dNTPs that are floating around to create this DNA strand, as well as the Magnesium Chloride to act as a catalyst. This then creates the two new strands of DNA, and this continues, causing the DNA strand to grow exponentially over time. The image only shows what occurs with the cancerous DNA, however. The normal DNA strand is also replicated, but nowhere near as rapidly as the cancerous one due to the primers that are in the solution not matching up with that of the non-cancerous strand of DNA. It replicates at a non-exponential rate, leaving a far larger abundance of the cancerous DNA throughout the process.