DNA Barcoding

DNA Barcoding

Kerr, A., Thom, W., Brennan, J.

Overview:

Biological Significance: DNA barcoding is essential to sequence DNA of a certain gene of unclassified organisms and match specific genes to databases to determine the species of the animal. When there is no match, barcoding can also show when a new organism has been found, and then that DNA sequence can be added to the database.

Methods:

In order to examine the DNA sequence of insects that were found in Poly Canyon, there are 4 main steps. DNA extraction is the first step to the process. This is used to extract isolate DNA from the nucleus of the cells. First, the cells were broken open, with the centrifuge, in a process called lysing, in order to release the cellular contents, including the nucleus. Then the cellular components were degraded other than DNA, by centrifugation and discarding of flow through after different rounds of buffers. Then the DNA was washed, with the DNA wash to remove any degraded components and other chemicals that were added during the DNA extraction process. This process leaves a sample of mostly pure DNA. Then the DNA is eluted, then extracted, and placed in a buffer.

The next main step is PCR, polymerase chain reaction. PCR is used to in the lab to make millions of copies of a specific piece of DNA, in this case it is the cytochrome c oxidase subunit I gene. The DNA is specified by the primer added into the extracted DNA sample. PCR requires the sample to be exposed to different temperatures, using a machine called a thermocycler. The amplified section of DNA is the mitochondrial cytochrome c oxidase subunit I gene common to many arthropods.

The next main step is gel electrophoresis, which makes it possible to visualize how successful the PCR process was of the DNA sampled, based on how similar and defined the samples are to the ladder, a solution of DNA molecules of different but known length used to determine length of the unknown DNA sample. Gel electrophoresis is used to visualize the success of PCR because the DNA sequences are too small to view under a microscope. The first step was preparing the gel, that will later be loaded with the replicated DNA samples into. The gel contains EtBr, a chemical which binds to DNA and it glows when exposed to UV light, making it possible to see the DNA. Both a positive, the ladder, and negative control, as well as 6 student samples were included. Then the gel was run, first an electrical current, at 100 volts, was run through the gel, which allows DNA fragments to travel through the gel. The distance in which each fragment travels depends on its size, the smaller the fragment the farther it will travel in the gel. From this the gel was removed from the buffer and placed in the UV light box in the Gel Documentation system. This visualizes and photographs a picture of the DNA in the gel, and how far each sample traveled. This machine is what exposes the DNA to UV light making it visible. Based on the picture, the relevant, the darkest, bands can be identified, these/this is the DNA which was the most successful in PCR of the DNA samples. The length is also determined by comparing the placement of the darkest band to the ladder.

The last component to this process was sending the sample corresponding to the most relevant band to be sequenced by a company. Once sequenced, the results were sent back, and the exact species and classification of the sample could be determined.

Results: - Attached are the PCR/Gel image results and the Barcoding Sequence Results

Discussion/Conclusions:

Part I:   There was only one band of DNA about halfway down the gel, meaning that the DNA was middle-sized in the amount of base pairs that it contained. This means that the PCR was effective because it made many copies of the one target DNA fragment, causing only one band to appear. The gel confirmed that the PCR worked and that the sample could effectively be used for further analysis such as DNA sequencing.

Part II: The data from the DNA of the insect matched the hypothesis regarding what type of insect it would be. The hypothesis stated that it would be an insect resembling a termite and the date summary confirmed the species is reticulitermes sp. which is a termite. The results summary also shows that specimens of the reticulitermes sp. have previously been found in California, just like this one.

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