Application of DNA Barcodes to Identify Various Plant Species
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 the ability of the BLAST technique to identify and distinguish plant species. Introduction
This experiment used the innovative technique of DNA barcoding. Barcoding is the process of using “short genetic sequence from a standard part of the genome to identify species. It works similarly with the way a supermarket scanner distinguishes products using the black stripes of the Universal Product Code (UPC). Two items may look very similar to the untrained eye, but in both cases the barcodes are distinct” (Hebert, 2003). According to Hebert, the most effective barcode regions in plants are found in the chloroplast (matK and rbcL). Our main goal here was to distinguish the various plant species from each other by “using” short orthologous DNA sequences (barcodes)” (Kress et.al, 2005). The differences in the DNA sequences allowed us to distinguish which species belong where and how they are related. The plant species identified were Spinach, Radicchio, Frisee, Endive, Red Lettuce, Green Lettuce Chard and a hypothesized Tatsoi (nonspinach). In this exercise, we expect to find the greatest similarity between the same plants, between members of the same species, between the species in one genus, between the genera of same family and between the same order. Methods
In this experiment, each group was given a bag of salad. Our group (B1 and B2) chose Chard as our specific plant type; however there were only two leaves present in the bag. As a result, my partner and I (group B1) shared one leaf. However, we sampled different sides of the leaf, in order to differentiate gene sequences (see Fig 1). Afterwards, we each isolated our DNA samples. We began by obtaining an isolation tube containing dilution buffer and crushed our plant sample in it using a 100 µl micropipette tip. We then took this mixture and spun it down in a centrifuge for 10 seconds to pellet the coarse plant fragments. Once we obtained the isolated DNA, we created a positive control and B2 created the negative control. After this was done, all the tubes were placed in a thermocycler. A week after, our samples were taken out of the thermocycler and the isolated samples were tested for amplification by electrophoresis. The wells of the agarose gel were loaded with our PCR sample and loading dye (see Table 1 for map). After the wells were loaded, we began electrophoresis, setting the voltage at 110V. We then ran the gel for one hour, around the time when the dye reached 3-4 cm. Then we transferred the gel into a staining tray and stained it with Ethidium Bromide for 15 minutes. After 15 minutes, the gel was observed under a UV light machine. A photograph was taken in order to analyze the results of DNA fragmentation. After the DNA has been amplified, it was purified. This was done using spin columns so the samples can be diluted. We accomplished the purification by adding Buffer PB and then observed the color changes in our sample. Since our sample immediately turned yellow, we moved on to its centrifugation and added buffer PE to wash the salts away. Buffer PE is made of ethanol and is used to wash the salt away from the DNA. We then added Buffer EB and centrifuged the sample again. DNA was quantified afterwards by using...
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