Dna Barcodes Lab

Title

Application of DNA Barcodes to Identify Various Plant Species

Abstract
In this experiment we applied barcodes to plants in order to identify what species they are classified under. We also compared the DNA sequences of different plant species using the ribulose-biphosphate carboxylase gene (rbcL). We took samples from a plant called Chard and performed PCR, DNA amplification and quantification and sequenced the DNA. During the experiment, we hypothesized that this year’s “nonspinach” is Tatsoi, however, our results proved otherwise upon completion of a BLAST (see Fig 9). The completion of a BLAST showed that “nonspinach” is actually spinach (Spinacia Oleracea) and our sample was indeed Chard (Beta vulgaris, Fig 10). This confirms
Dr. Raleigh had the samples sent out to a lab in MIT for sequencing, but we performed a simulation of the sequencing process using paper clips. We began by synthesizing complementary strands of paper clips based on the base-pairing rules and also the template strand provided. We made copies of the template strand by lining it up with a complementary sequence. We paired an unpaired base I in the template strand and paid attention to whether or not it was “deoxy” or “dideoxy.” We continued with this process until we have a) picked a nucleotide (paper clip) with its 3’ end taped or b) we have completed a complementary strand. We repeated the process until there were no free nucleotides left in the pool. We then sorted the chains by length (shortest to longest), leaving the template strand out. The data from the whole class was pooled and can be seen in Table 3.
A week later, Dr. Raleigh received the sequencing results and distributed the results to the corresponding group (see Figs 4-6). A comparison between the forward and reverse sequences was made and the sequence was trimmed so a “clean” sequence was obtained. A clean sequence refers to a sequence whose primer and ragged ends were detached. A family tree was then constructed in order to establish relationships between different species. Only one sample from our group was used since the rest did not have enough material, so the other groups’ results were utilized as
Figure 2 also indicated the success of the PCR amplification since we were able to construct the gel map found in Table 1. In Table 1, lanes 1, 3, 6, 9,12 and 14 were expected not to show any DNA since they were not loaded. We used different primers for the positive and negative controls for the sole of validity and accuracy of the 100bp ladder when comparing bands in the experimental lanes. Our positive control was designed to amplify 297 base pairs. We expected to find 1 fragment we did. On the other hand, we did not expect any fragments to show in the negative control, since it did not contain any DNA and we were successful. In the lanes containing experimental samples, we expected to find a single band for each lane indicating that only one DNA fragment was amplified. On the basis of our gel, we can conclude that we were successful in amplifying the DNA of Chard because all of our lanes came out as expected. The experimental samples appeared to be identical, which makes sense because we amplified the same rbcL gene sequence fragment, which suggests identical banding patterns. Based on our gel alone, we cannot tell plant samples apart, but merely if the correct DNA fragment was amplified. For this region of DNA however, we expect to see some subtle differences between other plant specimens since some plants have more than one primer-bonding region. The rbcL fragments amplified in our