Hope Hayman
DNA Bar Coding: Is Convenient and Accurate for Taxonomy Studies
Introduction— Different samples of “wild card” subjects and given subjects (Drosophila melanogaster and the Coquina clams) genomic DNA were acquired and isolated through several steps to amplify the cytochrome c oxidase 1 (CO1) genes. Through comparisons of these wild cards and given subjects mitochondrial CO1 genes with the BLAST library, it was revealed that DNA bar coding is convenient and accurate for taxonomy study. DNA bar coding utilizes the amplification and purification of a specific region of the mitochondrial genome by polymerase chain reactions (PCR). DNA bar coding then uses the PCR products ran in gel electrophoresis to analyze.
Materials and Methods—
DNA Isolation
The wild card specimens were obtained and brought to the lab for DNA isolation. The first step in DNA isolation was to lyse the specimen. The specimens were first homogenized individually in 1.5 mL microtubes. 20 µL of proteinase K was added to each homogenized sample. 200 µL of AL buffer was then added to each sample. The samples were vertexed and incubated at 70oC for 10 minutes. After the incubation, 200 µL of 100% ethanol was added and vortexed again. The next step in DNA isolation is to bind the DNA. The lysate was transferred to a spin column and centrifuged at 8000 rpm for 1 minute, and the flow through was discarded. 500 µL of AW1 buffer was added, then centrifuged at 8000 rpm for 1 minute, and the flow through was discarded. 500 µL of AW2 buffer was added and centrifuged at full speed for 3 minutes. Next in DNA isolation was the wash and elute phase. The spin column was put in a new 1.5 mL microtube. 100 µL of water was added to elute the genomic DNA. The specimens in the microtubes were left standing at room temperature for 1 minute. The samples were then centrifuged at 8000 rpm for 1 minute. The spin column was then discarded, and the microtube was kept. The flow through remaining in the tube is the genomic DNA; it was then stored at 4oC.
PCR (amplification of CO1 gene) and PCR Purification
The amplification of the mitochondrial CO1 gene was done using the following conditions and primers: LEPF1 and LEPR1 were the two primers. The samples were heated at 95oC for 2 minutes. The samples were then heated at 94 oC for 40 seconds, 51 oC for 40 seconds, and 68 oC for 70 seconds for 40 cycles. The samples were then heated to 68 oC for 5 minutess and stored at 4 oC.
Purification of the PCR products was done by centrifuging the samples for 5-10 seconds. The samples were then vortexed and centrifuged again for 5-10 seconds. The entire solution was transferred to a new 1.5 µL tube. 250 µL of PB buffer was added to the solution. 300 uL of the sample was transferred to a spin column and centrifuged at 13000 rpm for a minute. The flow through was discarded. 750 µL of PE was added to the solution and centrifuged at 13000 rpm for 1 minute. The flow through was discarded. The sample was centrifuged again at 13000 rpm for 1 minute; the spin column was transferred to a new 1.5 µL tube. 30 µL of water was added and let sit at room temperature for 1 minute. The sample was then centrifuged at 13000 for 1 minute. The column was discarded and the flow through contained the amplified PCR CO1 genes.
Agarose gel Electrophoresis and DNA Sequencing
10 µL of each sample was placed into 1.5 mL tubes. 3uL of loading dye was added to the 10 uL of purified DNA. The 13 µL of sample was then loaded into the agarose gel electrophoresis into different holding wells. The gel was run until the orange dye could be seen at about half way to two thirds of the way down the gel.
A portion of the samples were sent to Columbia University sequencing facility to provide a sequence of the amplified CO1 gene.
Results— The tested hypothesis was that DNA bar coding was accurate and convenient for taxonomy studies. This was tested to see how truly efficient this was at determining what the species were for samples of known organisms.
Table 1 shows the amount of successfully amplified CO1 DNA genes through the entire sample size. For the Drosophila mitochondrial CO1 DNA gene 18/39 samples were successfully amplified. For the Coquina mitochondrial CO1 DNA gene 4/39 samples had a successfully amplified CO1 gene. For the wild card mitochondrial CO1 DNA gene 16/20 successfully amplified the CO1 gene. Figure 1 shows the gel electrophoresis of the amplified mitochondrial CO1 DNA from Drosphila, Coquina, and the wild cards. Each gel electrophoresis came out differently. For the first two gels, they show the successfully amplified CO1 gene and almost all of the samples were successful. The next set of gels the success rate of amplifying the CO1 gene was a lot lower than the first two. Figure 2 shows some of the results of using BLAST on the obtained from sequences provided from Columbia University. The dog sequence provided by Columbia University was run through BLAST (Basic Local Alignment Search Tool), and the result concluded from the BLAST was that it was indeed a dog, Canis lupus familiaris. For the BLAST results of the provided sequence for the coquina, it was conclusive that it was a bivalve, Euheterodonta. The rabbit BLAST proved to be one of the only few that were different then what the provided sample said it was to be; the BLAST sequencing said the rabbit was a fly (Drosophila melanogaster).
Table 1: Success Rate of Amplified DNA | Drosophila Mitochondrail CO1 DNA (D) | Coquina Mitochondrial CO1 DNA (CA | Wild Card Mitochondrial CO1 DNA (W) | Success Rate | 18/39 | 4/39 | 16/20 |

Description: this table shows the amount of successful amplified CO1 DNA genes with the sample size.

Figure 1: Amplified Mitochondrial CO1 DNA

Description: This figure shows the gel electrophoresis of the amplified mitochondrial CO1 DNA from Drosphila, Coquina, and the wild cards.

Figure 2: Verification using BLAST
Dog:
Accession | Description | Max score | Total score | Query coverage | E value | Max ident | EU789778.1 | Canis lupus familiaris isolate A2_D9 mitochondrion, complete genome | 739 | 739 | 94% | 0.0 | 84% | DQ480507.1 | Canis lupus isolate 2 from Saudi Arabia mitochondrion, complete genome | 739 | 739 | 94% | 0.0 | 84% |

Coquina: Accession | Description | Max score | Total score | Query coverage | E value | Max ident | GU806100.1 | Euheterodonta sp. BOLD:AAJ1175 voucher MMI10 cytochrome oxidase subunit 1 (COI) gene, partial cds; mitochondrial | 143 | 143 | 76% | 2e-30 | 72% | GU806099.1 | Euheterodonta sp. BOLD:AAJ1175 voucher MMI8 cytochrome oxidase subunit 1 (COI) gene, partial cds; mitochondrial | 130 | 130 | 76% | 1e-26 | 72% |

Rabbit (resuLts differ from the given specimen): Accession | Description | Max score | Total score | Query coverage | E value | Max ident | GQ229519.1 | Drosophila melanogaster strain Oregon R cytochrome c oxidase subunit I (CoI) mRNA, partial cds; mitochondrial | 1221 | 1221 | 99% | 0.0 | 99% | FJ190105.1 | Drosophila melanogaster isolate w1118iso mitochondrion, partial genome | 1221 | 1221 | 99% | 0.0 | 99% |

Description: This shows some of the results of using BLAST on the obtained from sequences provided from Columbia University.

Discussion— The success rate of the amplified CO1 DNA seemed to be very low. This might have been due to the lack of experience in handling the procedure and equipment or that the there was an error in the provided primers and/or buffers that were provided (as seen in Table 1 and the gels). This tested the convenience and accuracy of DNA bar coding using the materials provided. Due to the lack of the success rate of the amplified mitochondrial CO1 genes and how some of the sequences provided for the “wild cards” did not match up with the known subject’s name, it was led to believe that the hypothesis partially supported. Although this method is convenient in producing sequences that are able to be utilized fast, it is not always accurate at producing the right taxonomy for the subjects. This could be due to contamination, mislabeling, or bad methods being used. Doing this experiment over with more experienced lab technicians might produce different and more accurate results for both the gel results and the BLAST results. Perhaps if the future lab technicians become more familiar with the lab kits provided and use them more frequently these problems could be solved.