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PCR is the process of using primers to copy DNA. Primers are DNA single-stranded segments of 20-25 base pairs that are complimentary to DNA strands. They bind to the DNA at areas where the base pairs are complimentary and copy the DNA strand from 5' to 3'. PCR is run in a machine called a thermal cycler that regulates the temperature. The first temperature is usually between 94 and 96 degrees Celsius and is to break the bonds in DNA. This cycle usually last 2-10 minutes. The second step in the denaturing step (from 94-96 degrees for 30 seconds); this step separates the two strands of DNA. The next step is the annealing step (50-55 for about one minute); this is the step where primers bond to both strands of DNA from 5' to 3'. There is an extension at 72 degrees Celsius, which extends the chain by copying the strand. Denaturing, annealing, and extension are then repeated 25-35 times before bringing the sample down to room temperature and ending the program. The annealing temperature is set according to the primer, and is usually 3 or 4 degrees below the highest melting point of the primer, which is determined by the GC (Guanine and cytosine, two of the four nucleic acids that make up DNA) content. The components of PCR are the DNA template, the forward and reverse primers, the DNA polymerase, a buffer, dNTP, and water. The DNA template is isolated DNA from the last step in our process. We use a 2x premade mastermix for PCR made by Bio-rad that contains the DNA polymerase, buffer, and dNTP. All we have to add is DNA, primers, and water to get the solution to the correct concentration. We use purified water specifically made for PCR because the water can't contain any other DNA that could be amplified. We started by using a 16srRNA primer, a universal primer that amplifies a gene that can be used to identify the species of bacteria. We use the following recipe for our 16s PCR reactions:
We ran this recipe with the following thermal cycler program:
Once we successfully amplified the 16s gene and found that it was similar to several species of Thermus, including Thermus Thermophilus, Thermus Aquaticus, and Thermmus Brochianus, we used already sequenced genes of these species to design our own primers for our bacteria's polymerase gene. The polymerase gene of Thermus Aquaticus is the main polymerase gene used in PCR for all reactions. It is useful because of its resistance to heat. Therefore, our polymerase gene may also be useful in PCR. The program we used for primers designed for Brochianus is:
The program we used for primers designed for Aquaticus and Thermophilus is:
Once we complete PCR, our next step is to run the products out on agarose gels in a process called gel electrophoresis. This allows us to determine if our primers worked and our PCR was done correctly. We can determine whether we amplified the correct gene by the size of the DNA (where it lies in the gel). |
