As mentioned last week, I have been spending some time perusing the 16s gene sequences of my target organisms and was able to design a number of primers for use in PCR. The primers have been ordered, and that portion of my research is in a holding pattern until the order arrives.
In the meantime, I have been testing Universal Rice Primers (URPs) for universality against the eight bacterial species I am studying. URPs are called universal arbitrary primers because, rather than targeting specific gene sequences without variation, they adhere to and amplify random nucleotide sequences during the PCR process. Theoretically, this allows them to be used with a greater variety of organisms than the sequence-specific primers I am testing. However, in my first attempt to amplify DNA using these primers and a protocol previously used by a former S-STEM student, my PCR was unsuccessful. I surmised that this was likely due to the lack of necessary data from the previous student's experiment, as all I had to go on when attempting replication of the experiment was the poster that had been created with a synopsis of the experiment. Detailed, relevant information, such as the concentration of the primer dilution used, was missing.
In order to surmount this obstacle, I devised an experiment that utilized repetition and manipulation of variables to potentially identify the reason(s) for the failure of the first experiment. Using the same DNA samples that were utilized in the first test, I changed the annealing temperature of the PCR protocol to a lower temperature than previously used; I also repeated the original primer dilution but added another dilution factor as a variable to test concentration effect on PCR results. Given the variables being tested, I had a total of forty-eight samples being tested in one experiment.
The results? DNA amplification successful! Data analysis indicated that primer concentration did affect PCR results, as there were variances in banding frequency and size when the electrophoresis gels were reviewed post-PCR. However, I determined that temperature was the primary factor for the differences in results between experiments #1 and #2; given that I repeated all of the original controls, changed only the annealing temperature, and left the original primer concentration intact for the second experiment, this indicated that temperature was the culprit in the first experiment.
The addition of a primer concentration variable to the experiment did provide valuable data. Gel analysis revealed that DNA banding was less prevalent at the lower concentration for all but one of the primers, which instead showed an increase in banding clarity. This result will help me in designing future experiments using these URPs, as it revealed the optimal dilution concentration formula for primers tested at the annealing temperature selected.
Next week, I will be repeating the experiment using primers of my own design. Until then, in the words of S'chn T'gai Spock, "Live long and prosper."
And speaking of rice, here is an interesting video on GMO'd golden rice.
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