Introduction
The question of how to define what a species is has been a contentious issue for as long as the concept has been around. Even without a consensus on definitions the idea of a species is fundamental to all fields of biology, especially so with evolutionary biology. The term “I know it when I see it” can be a good way to define a species, however, while historically morphology has been used to classify organisms into species, mimicry as well as the tediousness and expertise required to fully describe an organism does not allow for the widespread adoption of morphology to accurately describe the natural world (Kasap, Linton, Karakus, Ozbel, & Alten, 2019). The switch from looking at phenotype to genotype when classifying species can be done via DNA barcoding. Marker genes are utilized for this purpose to act as a “barcode” for a species. Genes that have high interspecies variation and low intraspecies variation are what are used as marker genes (Wang, Wen, Edihara, & Li, 2016). For photosynthetic organisms the gene rbcL is used to barcode (Wang et al., 2016). rbcL codes for ribulose-1,5-bisphosphate carboxylase (RuBisCo) an enzyme ubiquitous in plants by such a degree that it may outnumber all other proteins on Earth (Wang et al., 2016). Once rbcL, or any other gene used as a barcode is extracted and amplified the sequence can then be determined and compared with other gene sequences to determine an organism’s relationship to others quantitatively (Newmaster, Grguric, Shanmughanandhan, Ramalingam, & Ragupathy, 2013).
[bookmark: _Hlk23409206]DNA Barcoding is not just of interest to biologists trying to construct a phylogenetic tree but to other areas of interest as well. Conservation, epidemiology, and food and drug quality control are just several the fields that utilize DNA barcoding to understand and address issues in their fields. Regarding conservation, the problem of using species concepts such as morphology or the biological species concept, the concept that defines species based on the ability to produce viable offspring, is that many organisms, particularly plants, can develop viable offspring via interspecies reproduction thereby producing a novel species thereby complicating the process (Wang et al., 2016). DNA barcoding allows for a relatively rapid and accurate identification of plants (Zhang et al., 2019). Somewhat related is the usage in epidemiology as the tracking of vectors that carry diseases can be done at a far greater capability than by any other means (Kasap et al., 2019). Unscrupulous additives in food and supplements are also an area of concern as the regulations regarding listing ingredients was found to be ill enforced (Zhang et al., 2019). DNA barcoding can be used by monitoring agencies to regulate and enforce accurate listings of ingredients and deter fraudulent conduct (Newmaster et al., 2013) (Zhang et al., 2019).
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To test the efficacy of DNA barcoding in what was believed to be blueberries from Kroger®, referred to as test sample, were processed to extract DNA. PCR was conducted to amplify the blueberries rbcL gene. The Amplified PCR products were then purified via washing. Gel electrophoresis was conducted to determine successful amplification of the rbcL gene. Extracted DNA and the Unpurified DNA were used as negative and positive controls respectively. The Purified DNA was the test sample. Sequences were submitted to BLAST and analyzed. The goal of this experiment is to determine the identification of the test sample utilizing DNA barcoding.
Materials and Methods
I. DNA Extraction and PCR
15 mg of the test sample was ground thoroughly. 300 µl of nucleic lysis solution was added to the ground sample. The resulting slurry was further ground thoroughly. The final ground test sample/nucleic lysis solution slurry was incubated at 65 ℃ for 10 minutes. Following incubation, the slurry was centrifuged at 14,000 rpm for 4 minutes. 150 µL of the supernatant from was transferred to a new tube where then 10 µL of silica gel was mixed with the supernatant. The tube was incubated at 57 ℃ for 15 minutes. The tube was then centrifuged at 14,000 rpm for 30 seconds where then the supernatant was discarded. 500 µL of ice-cold wash buffer was added to the pellet. Centrifugation at 14,000 rpm for 30 seconds was repeated, as was the removal of the supernatant, and washing with 500 µL of ice-cold wash buffer. Another centrifugation was conducted at 14,000 rpm for 30 seconds. The supernatant was discarded, and 100 µL of molecular biology grade distilled water was added. The was pellet resuspended via vortexing . This was followed by incubation at 57 ℃ for 5 minutes and centrifugation at 14,000 rpm for 30 seconds. 90 µL of the supernatant was transferred to a new tube.
23 µL of an Orange G loading dye and rbcL primer solution was added to a PCR tube that contained Ready-To-Go PCR Beads™. 2 µL of the supernatant from the extraction procedures was transferred to the PCR tube. 88 µL of the remaining supernatant was saved for gel electrophoresis and shall be referred to as Extracted DNA (D). PCR was conducted with the following thermocycler program: 94 ℃ for 15 seconds for denaturation, 54 ℃ for 15 seconds for annealing, and 72 ℃ for 30 seconds for extension. The cycle of Denaturation, annealing, and extension was repeated for 35 cycles after which it was held at 4 ℃. The PCR product was then stored at -20 ℃.
II. DNA Purification and Gel Electrophoresis
20 µL of the PCR product was transferred to a 1.5 mL centrifuge tube. The remaining 5 µL was saved for gel electrophoresis and shall be referred to as Unpurified DNA (U). 30 µL of water was added to the 1.5 mL centrifuge tube followed by an addition of 250 µL of buffer PB to the solution. This was centrifuged at 7,000 rpm for 30 seconds. The solution in the 1.5 mL tube was transferred to the upper chamber of a QIAquick spin column. This was centrifuged at 14,000 rpm for 1 minute. The filter was removed, and the collection tube was emptied before the spin column was reassembled. 750 µL of buffer PE was added to the upper chamber of the spin column. This was centrifuged at 14,000 rpm for 1 minute. The filter was removed, and the collection tube was emptied before the spin column was reassembled. To dry the spin column was centrifuged at 14,000 rpm for 1 minute. The collection tube was discarded and replaced. 50 µL of elution buffer was added to the spin column. The spin column was centrifuged at 7,000 rpm for 3 minutes. The collection tube was saved for gel electrophoresis and shall be referred to as Purified DNA (P).
The gel was made with 1g of agarose to 100mL of 1X TAE buffer. 10 µL of Purified DNA, 5 µL of Unpurified DNA, and 10 µL of Extracted DNA were each prepared with 4.5 µL of ethidium bromide and 3 µL of deionized water. Electrophoresis was conducted at 170 V for 25 minutes
Results
Three of four wells show bands. Well 1 contains the Carolina pBR322/BstNI molecular weight ladder which shows four bands at 1058bp, 929 bp, 383 bp, and 121 bp. Well 2 is Purified DNA and shows a band (P band) between 929 bp and 383 bp. Well 3 is the Unpurified DNA and shows a band (U band) between 929 bp and 383 bp, the band is slightly higher than the P band. Well 4 shows no band and is the Extracted DNA. Wells 2 and 3 contain the amplified rbcL marker gene and are the test sample and positive control respectively. Well 4 contains no amplified DNA of any kind and is the negative control.
Works Cited
- Kasap, O. E., Linton, Y.-M., Karakus, M., Ozbel, Y., & Alten, B. (2019). Revision of the species composition and distribution of Turkish sand flies using DNA barcodes. Parasites & Vectors, 12(1). doi: 10.1186/s13071-019-3669-3
- Newmaster, S. G., Grguric, M., Shanmughanandhan, D., Ramalingam, S., & Ragupathy, S. (2013). DNA barcoding detects contamination and substitution in North American herbal products. BMC Medicine, 11(1). doi: 10.1186/1741-7015-11-222
- Wang, F.-H., Lu, J.-M., Wen, J., Ebihara, A., & Li, D.-Z. (2016). Applying DNA Barcodes to Identify Closely Related Species of Ferns: A Case Study of the Chinese Adiantum (Pteridaceae). Plos One, 11(9). doi: 10.1371/journal.pone.0160611
- Zhang, M., Shi, Y., Sun, W., Wu, L., Xiong, C., Zhu, Z., Zhao, H., Zhang, B., Wang, C., & Liu, X. (2019). An efficient DNA barcoding-based method for the authentication and adulteration detection of the powdered natural spices. Food Control, 106. doi: 10.1016/j.foodcont.2019.106745
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