The Development Of Primers For Unknown Thermophilic Bacteria At Vulcan Hot Springs

1723 words - 7 pages

The Thermus Aquaticus project’s purpose is to identify a species of thermophilic bacteria found in a local hot spring, Vulcan Hot Springs, through genetic sequencing and analysis. The polymerase gene in other Thermus bacterias has proven useful in genetic reactions. The Vulcan bacteria grows at a higher temperature than other thermophilic bacterias, giving it the potential to have a more effective polymerase gene than what is currently available. My own research has been focused on designing effective primers for the polymerase gene in the bacteria. I have designed primers based on the similarities between the Vulcan bacteria and known Thermus species found in GenBank after growing, isolating, running polymerase chain reaction (PCR), running gel electrophoresis, and sequencing samples in the labs at Cascade High School and Idaho National Laboratories. I have not been able to obtain consistent and correct results from these primers.

INTRODUCTION
Cascade High School’s (in Cascade, Idaho) research lab projects are student taught – passed down from one student to the next each year. Thermus Aquaticus (TAQ) is a research lab project focused on identifying a thermophilic bacterium found in a local hot spring, Vulcan Hot Springs. The polymerase gene in bacteria species, mainly Thermus aquaticus, has proven useful in polymerase chain reactions (PCR), an important reaction in genetic and forensic sciences. The Vulcan bacterium grows at a higher temperature than Thermus aquaticus; the polymerase gene from Vulcan may prove to be more useful than those currently available.

I joined TAQ as a sophomore in high school and learned much of the project, especially the procedures completed on a computer, on my own. After sequencing the 16SrRNA gene of the Vulcan bacterium and comparing this sequence to those of other thermophilic bacteria found in GenBank, I designed primers based on the similarities between the species. My personal focus in TAQ is to design effective primers for the Vulcan bacterium using CLC DNA Workbench, Primer3, and GenBank. Not having anything but basic computer knowledge, I faced many issues with the software programs, including interpreting seemingly futuristic language and symbols.

METHODS
Several steps must be completed to prepare the bacterium DNA for sequencing. The first step is extraction of bacteria cultures from Vulcan Hot Springs. Samples were collected from various areas in Vulcan Hot Spring and brought to the lab at Cascade High School. The Vulcan bacterium grows at 75 degrees Celcius in a hot water bath with the shaker on. After the samples are growing well, isolation can be run. Isolation, or extraction, is the process of removing the DNA from the cells of the bacteria in order to perform more reactions on the DNA. This process rids the samples of unneeded materials. After isolation, a polymerase chain reaction is run on the samples in order to amplify the specific gene needed. Following PCR, either a gel is run to...

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