DNA Barcoding Primer Design for Species Identification of Skipjack Tuna, Yellowfin Tuna, and Mackerel: Development and Validation of Primers for Genetic Research
DOI:
https://doi.org/10.15408/kauniyah.v19i1.42706Keywords:
Tongkol, Tuna Sirip Kuning, Cakalang, DNA Barcoding, Desain Primer, PCR (Polymerase Chain Reaction)Abstract
The DNA barcoding technique is primarily utilized to rapidly identify species, particularly when samples are damaged and cannot be identified accurately through morphological methods. This approach utilizes polymerase chain reaction (PCR) technology to amplify DNA fragments from the targeted species, with its success largely due to the design of the primers employed. The cytochrome c oxidase subunit I (COI) gene, a mitochondrial gene, is frequently targeted in DNA barcoding and has been proven effective in distinguishing species. At Pondokdadap Port, over 90% of the fish caught consist of skipjack tuna, yellowfin tuna, and mackerel (T2C). This study aimed at designing silico DNA barcoding primers for these three species. The successful development of these primers may facilitate the documentation and understanding of the genetic diversity of the species under study, which is crucial for efficient and effective fisheries management. The primer design process applied Primer-BLAST software from the NCBI website, followed by additional testing with OligoAnalyzer. The selected primer pairs were the forward primer 5'-GGCCCATGCCTTCGTAATGA-3' and the reverse primer 5'-GCAGGGTCGAAGAAGGTTGT-3'. These primers successfully amplified the DNA of T2C fish, with PCR results indicating that the optimal annealing temperature for these primers was 55 °C
References
Agustina, M., Jatmiko, I., & Sulistyaningsih, and R. K. (2020). Ulur Tuna Di Perairan Sendang Biru Catch Composition and Fishing Ground of Tuna Handline. Jurnal Perikanan Indonesia, 25(4), 241–251.
Anika, M., Putri, D. H., & Wahyuni, W. (2019). Primer design for identification of beta-carotene encoding genes in cassava. Bio sains, 4(1), 39-47.
Barbeyrac, B. D. & Bebear, C. (1996). Molecular and diagnostic procedures in mycoplasmology (second edition). Elsevier.
Doorenweerd, C., Jose, M. S., Leblanc, L., Barr, N., Geib, S., Chung, Arthur Y.C., Dupuis, J., Ekayanti, A., Fiegalan, E. R., Hemachandra, K.S., Hossain, M. A., Huang, C. L., Hsu, Y. F., Morris, K. Y., Mustapeng, A. M., Niogret, J., Pham, T. H., Sirisena, U. G. A. I., & Rubinoff, D. (2020). DNA barcodes and reliable molecular identifications in a diverse group of invasive pests: lessons from Bactrocera fruit flies on variation across the COI gene, introgression, and standardization. BioRxiv, https://doi.org/10.1101/2020.11.23.394510.
Folmer, O., M. Black, W. Hoeh, R. Lutz, R. Vrijenhoek. (1994). DNA Primers for Amplification of Mitochondrial Cytochrome C Oxidase Subunit 1 from Diverse Metazoan Invertebrates. Mol. Mar. Bio. Biotech., 3, 294–299.
Kim, S. H., Lee, M., Choi, K. H., Jeong, J., Lee, D. K., Oh, J. N., Choe, G. C., & Lee, C. K. (2022). Species-specific enhancer activity of oct4 in porcine pluripotency: the porcine oct4 reporter system could monitor pluripotency in porcine embryo development and embryonic stem cells. Stem Cells International, 2022, 1-18.
Pasha, F., Saggu, S., & Albalawi, M. F. (2016). Future perspectives of DNA barcoding in marine zooplanktons and invertebrates. In DNA Barcoding in Marine Perspectives: Assessment and Conservation of Biodiversity. https://doi.org/10.1007/978-3-319-41840-7_11.
PPP Pondokdadap. (2020). Mengakhiri bulan agustus 2020, produksi ikan pelagis kecil melonjak tajam di UPT PPP Pondokdadap. Retrieved from https://infopondokdadap.com/mengakhiri-bulan-agustus-2020-produksi-ikan-pelagis-kecil-melonjak-tajam-di-upt-ppp-pondokdadap/
Pradnyaniti, D., Wirajana, I., & Yowani, S. (2014). Desain Primer secara in silico untuk amplifikasi fragmen Gen rpoB Mycobacterium tuberculosis dengan Polymerase Chain Reaction (PCR). Jurnal Farmasi Udayana, 2(3), 124–130.
Sengal, A. T., Mohamedani, A. A., Hussein, H. H., & Kamal, A. (2016). The role of pcr in diagnosis of a rare appendicular tuberculosis and mini literature review. Case reports in gastrointestinal medicine, 2016, 1-3.
Setiyawan, S. A., Budiharjo, A., & Kusumaningrum, H. P. (2015). Seleksi Primer LCO – HCO, Primer bird-f1 – HCO Dan Primer bch – bcl Untuk Amplifikasi Gen COI DNA Mitokondria Itik Magelang (Anas javanica). Bioma : Berkala Ilmiah Biologi, 16(2), 83. https://doi.org/10.14710/bioma.16.2.83-89.
Setyawati, R. & Zubaidah, S. (2021). Optimasi konsentrasi primer dan suhu annealing dalam mendeteksi gen leptin pada sapi peranakan ongole (po) menggunakan polymerase chain reaction (PCR). Indonesian Journal of Laboratory, 4(1), 36-40.
Sharma, P. & Kobayashi, T. (2014). Are “universal” DNA primers really universal?. J Appl Genetics, 55, 485–496. https://doi.org/10.1007/s13353-014-0218-9.
Sihotang, M. A. E. D., Erwinda, Y. E., Suwarni, E., & Lusianti, E. (2021). Desain Primer dan Analisis in Silico untuk Amplifikasi Gen mt-Co1 pada Tikus got (Rattus norvegicus). Eruditio : Indonesia Journal of Food and Drug Safety, 1(2), 20–29. https://doi.org/10.54384/eruditio.v1i2.82.
Suparman, Suparman; Ahmad, Hasna; Ahmad, Z. (2016). Desain Primer PCR Secara In Silico untuk Amplifikasi Gen COI pada Kupu-Kupu Papilio ulysses Linnaeus dari Pulau Bacan. Jurnal Pendidikan Matematika Dan IPA, 7(1), 14–24. https://doi.org/10.26418/jpmipa.v7i1.17341.
Wu, T. H., Yang, C. H., Pai, T. W., Ho, L. P., Wu, J. L., & Chou, H. Y. (2021). Identification of fish species through tRNA-based primer design. BMC Bioinformatics, 22(2021), 1–15. https://doi.org/10.1186/s12859-022-04717-8.
Yuenleni, Y. (2019). Langkah-langkah optimasi pcr. Indonesian Journal of Laboratory, 1(3), 51-56.
