OPTIMASI DETEKSI GEN PADA Stelechocarpus burahol (Bl.) Hook.f. & Th. MENGGUNAKAN DIRECT KIR PCR

Tri Suwarni Wahyudiningsih; Dian Sartika

doi:10.20886/jpth.2020.14.2.93-99

OPTIMASI DETEKSI GEN PADA Stelechocarpus burahol (Bl.) Hook.f. & Th. MENGGUNAKAN DIRECT KIR PCR

Tri Suwarni Wahyudiningsih, Dian Sartika

Abstract

Purification of DNA molecules from a large number of samples is laborious, costly, time-consuming, and a high risk of contamination. S. burahol leaves contain phenolic compounds, flavonoids, and terpenoids that can interfere with DNA isolation. Direct PCR kits can detect genes without DNA extraction. The objective of study was to determine the method of gene detection of Stelechocarpus burahol using the direct PCR kit. S. burahol leaf samples came from the Bogor Botanical Gardens (two trees), Garut, Purwodadi Botanical Gardens, Kyai Langgeng Gardens, Yogyakarta Palace, Turi Sleman, Wanagama, Karanganyar, and South Kalimantan. Each leaf sample of 0.1 mg was dissolved into 1.25% w / v SDS (Sodium Dodecyl sulfate) 50 ul solution, incubated at 95^o C for 5 minutes, and vortexed for 2 seconds. Primers used for the trials were ITS 1F primers and 4R primers. Pre-denaturation of 95^oC for 7 minutes, denaturation of 95^oC for 1 minute, annealing 55^oC for 1 minute, extension at 72^oC for 1 minute, extension at 72^oC for 1 minute, and 40 cyclus. PCR product samples of 40 - 50 μl that showed positive results were detected by electrophoresis. The results of S. burahol DNA amplification measuring ± 750 bp from ten samples of S. burahol. Direct PCR kits can be used for S. burahol gene detection, time and energy efficient, only requires a small amount of tissue, and reduces contamination due to DNA extraction. Direct PCR kits can be an effective method that can be utilized to detect target genes for large populations

Keywords

detect target genes; DNA amplification; PCR product, without DNA extraction

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References

AlShahni M.M., Makimura K, Yamada T., Satoh Y. (2009). Direct colony PCR of several medically important fungi using Ampdirect® Plus. Japanese Journal of Infectious Diseases.

Aziz SA and BC Ramadhan. 2013. Media and organic fertigation for growth and phytochemical properties of Stelechocarpus burahol in nursery. International Seminar Proceedings Forests & Medical Plants for Better Human Welfare. Bogor, 10-12 September 2013.Hlm. 200-204.

Bellstedt, D. U., Pirie, M. D., Visser, J. C., de Villiers, M. J., & Gehrke, B. (2010). A rapid and inexpensive method for the direct PCR amplification of DNA from plants. American Journal of Botany. https://doi.org/10.3732/ajb.1000181

Ben-Amar, A., Oueslati, S., & Mliki, A. (2017). Universal direct PCR amplification system: a time- and cost-effective tool for high-throughput applications. 3 Biotech. https://doi.org/10.1007/s13205-017-0890-7

Ben Amar, A., Oueslati, S., Ghorbel, A., & Mliki, A. (2012). Prediction and early detection of mycotoxigenic Fusarium culmorum in wheat by direct PCR-based procedure. Food Control. https://doi.org/10.1016/j.foodcont.2011.08.021

Berthomieu, P., & Meyer, C. (1991). Direct amplification of plant genomic DNA from leaf and root pieces using PCR. Plant Molecular Biology. https://doi.org/10.1007/BF00040656

Cao, M., Fu, Y., Guo, Y., & Pan, J. (2009). Chlamydomonas (Chlorophyceae) colony PCR. Protoplasma. https://doi.org/10.1007/s00709-009-0036-9

Cascella, R., Strafella, C., Ragazzo, M., Zampatti, S., Borgiani, P., Gambardella, S., Pirazzoli, A., Novelli, G., & Giardina, E. (2015). Direct PCR: A new pharmacogenetic approach for the inexpensive testing of HLA-B∗57:01. Pharmacogenomics Journal. https://doi.org/10.1038/tpj.2014.48

Chum, P. Y., Haimes, J. D., André, C. P., Kuusisto, P. K., & Kelley, M. L. (2012). Genotyping of plant and animal samples without prior DNA purification. Journal of Visualized Experiments. https://doi.org/10.3791/3844

Eszik, I., Lantos, I., Önder, K., Somogyvári, F., Burián, K., Endrész, V., & Virok, D. P. (2016). High dynamic range detection of Chlamydia trachomatis growth by direct quantitative PCR of the infected cells. Journal of Microbiological Methods. https://doi.org/10.1016/j.mimet.2015.11.010

Hwang, H., Bae, S.-C., Lee, S., Lee, Y.-H., & Chang, A. (2013). A Rapid and Simple Genotyping Method for Various Plants by Direct-PCR. Plant Breeding and Biotechnology, 1(3), 290–297. https://doi.org/10.9787/pbb.2013.1.3.29

John, M. E. (1992). An efficient method for isolation of RNA and DNA from plants containing polyphenolics. Nucleic Acids Research. https://doi.org/10.1093/nar/20.9.2381

Kim, C. S., Lee, C. H., Shin, J. S., Chung, Y. S., & Hyung, N. I. (1996). A simple and rapid method for isolation of high quality genomic DNA from fruit trees and conifers using PVP. Nucleic Acids Research. https://doi.org/10.1093/nar/25.5.1085

Klimyuk, V. I., Carroll, B. J., Thomas, C. M., & Jones, J. D. G. (1993). Alkali treatment for rapid preparation of plant material for reliable PCR analysis. The Plant Journal. https://doi.org/10.1111/j.1365-313X.1993.tb00169.x

Li, F. W., Kuo, L. Y., Huang, Y. M., Chiou, W. L., & Wang, C. N. (2010). Tissue-direct PCR, a rapid and extraction-free method for barcoding of ferns. Molecular Ecology Resources. https://doi.org/10.1111/j.1755-0998.2009.02745.x

Nishimura N, Nakayama T, Tonoike H, Kojima K, Kato S (2000) Direct polymerase chain reaction from whole blood without DNA isolation. Ann Clin Biochem 3

Pathmanathan SG, Cardona-Castro N, Sa´nchez-Jime´nez MM, CorreaOchoa MM, Puthucheary SD, Thong KL (2003) Simple and rapid detection of Salmonella strains by direct PCR amplification of the hilA gene. J Med Microbiol 52:773–778

Purwantiningsih, I Purwantini dan D Santoso. 2011. Identification of standard parameters of kepel leaves [Stelechocarpus burahol (Bl.) Hook. F. & Th.] and the extract as raw material for antihyperuricemic medicaments. Asian Journal of Pharmaceutical and Clinical Research 4(1): 149153.

Sharma, R., Kumar, V., Mohapatra, T., Khandelwal, V., & Vyas, G. K. (2012). A Simple and Non-destructive Method of Direct-PCR for Plant Systems. Journal of Plant Biology. https://doi.org/10.1007/s12374-011-9191-6

Shokralla, S., Singer, G. A. C., & Hajibabaei, M. (2010). Direct PCR amplification and sequencing of specimens’ DNA from preservative ethanol. BioTechniques. https://doi.org/10.2144/000113362

Surzycki, S., & Surzycki, S. (2000). Preparation of Genomic DNA from Plant Cells. In Basic Techniques in Molecular Biology. https://doi.org/10.1007/978-3-642-56968-5_3

Werblow, A., Flechl, E., Klimpel, S., Zittra, C., Lebl, K., Kieser, K., Laciny, A., Silbermayr, K., Melaun, C., & Fuehrer, H. P. (2016). Direct PCR of indigenous and invasive mosquito species: A time- and cost-effective technique of mosquito barcoding. Medical and Veterinary Entomology. https://doi.org/10.1111/mve.12154

White, T.J., Bruns, T., Lee, S.J.W.T., dan Taylor, J.W. 1990. Amplification and Direct Sequencing of Fungal Ribosomal RNA Genes for Pylogenetics. PCR Protocol: A Guide to Methods Application. 18:315-322.

Young, G. Y., Jong, Y. K., Soh, M. S., & Kim, D. S. (2007). A simple and rapid gene amplification from Arabidopsis leaves using AnyDirect system. Journal of Biochemistry and Molecular Biology. https://doi.org/10.5483/bmbrep.2007.40.3.444

DOI: https://doi.org/10.20886/jpth.2020.14.2.93-99