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Sequence to Success DNA Barcoding Challenge Angelica Ruedas, Glenn Guardamondo, David Rodriguez, Ifechukwu Okeke and Ceasar Hurtado University of California, Riverside and Chaffey College Abstract Results / Analysis Fishes can be compared to other fish with the same DNA. The 16S ribosomal gene and cytochrome-­‐oxidase subunit (COI) are two parts to fishes’ DNA that are specific for all species of fish. To begin, the fishes’ DNA was extracted. Ader extracHon, polymerase chain reacHon (PCR) was done to target the specific sequence. To ensure that PCR had taken place, gel electrophoresis was done to allow the DNA bands to diffuse through the agarose gel; the heavier, copied bands moved at different rates than the rest of the DNA that was not targeted. The sequences were then sent, and ader receiving results from the sequence company, the DNA was compared to the sequences of the species of fish that resembled the DNA sequence of the extracted samples. For gel electrophoresis, the results showed that PCR had occurred. Sixteen out of twenty-­‐nine fish were incorrectly labeled by grocery stores. With this study we see fishes sold in local grocery stores were labeled incorrectly fidy-­‐five percent of the Hme. Background DNA barcodes allow idenHficaHon of individual fish due to part of the genes staying the same among fish species, and part of the gene being highly variable causing the fish to express different traits and look different. The sequence targeted in this case is the common gene on the DNA of all fish which codes for the 16S ribosome and this is called “mitochondrial targeHng”. The only ribosomes in the fish which are 16S are that of mitochondria, which were formerly prokaryotes, but became a part of the fish genome by endosymbiosis. Another region of the gene common in all fish is the cytochrome-­‐oxidase subunit I (COI) also found in the mitochondria of fish and has parts which stay stable for all fish species and parts which highly vary. Specific primers are applied to the region which are the same, so the variable regions become more obvious. To these variable regions, the polymerase chain reacHon is carried out so that they now become more amplified. A DNA strand containing the target sequence has primers added to it, and then heaHng is then done in order to denature and separate the strands which are then cooled to allow the primers bind properly to the ends of the strands, which are the common, unneeded regions. • 
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Methods DNA ExtracHon • 
Figure 1-­‐ PhylogeneHc tree derived from DNA Subway using the blue line. Figure 2-­‐The gel electrophoresis showed PCR targeted the specific sequence of ribosomal DNA that is unique to each fish. Extracted DNA from different fish samples Figure 3-­‐ The highlighted rows are those types of fish species that have been discovered to be different than what they were originally sold as. Conclusions and Future DirecHons Polymerase Chain ReacHon 16S primers were used to amplify the COI mitochondrial DNA • 
Gel Electrophoresis • 
Amplified DNA samples were run through a 1.5% agarose gel •  16 out of the 29 fish were in fact different species than the one specified by local markets. •  This has shown that more than half of the fish purchased in local markets were labeled and sold incorrectly. •  The following future direcHons can be pursued to beVer understand the miss-­‐labeling of fish: -­‐ Test fish samples focusing on what country they come from -­‐ Test fish samples from different fish farming companies to see which are the most reliable DNA Analysis DNA was sequenced using the Big Dye Cycle sequencing method. Acknowledgements Neil A. Campbell Science Learning Laboratory We acknowledge the following individuals for their assistance in our research: Dr. Richard Cardullo, Dr. James BurneVe, Mr. Alejandro Cortez, Dr. Darcie Descalzo, Mr. Nhi Tran, Dr. Venki CheVy, Ms. Michelle Butler, and Ms. Marissa Carranza