DNARS: A safe, environmentally friendly and high-quality DNA extraction method suitable for various biological samples

Cesar David Guerra Naranjo, Tanira Alessandra Silveira Aguirre, Ana Paula Guedes Frazzon

Resumen


The functional and interactional relationships between nucleic acids and proteins form the foundational framework for molecular biology studies. In this context, obtaining high-quality nucleic acids is a crucial step for successful downstream applications. This study aimed to develop a cost-effective, eco-friendly, and versatile DNA extraction method that yields high-quality DNA from diverse biological samples. We utilized three types of borosilicate-based recycled laboratory glass as a silica source, combined with sodium iodide as a chaotropic agent, creating an efficient system for cell lysis and DNA extraction.

Quality control was performed by assessing the concentration and purity of the extracted DNA using spectrophotometry, and the results were compared to those obtained with a commercial kit. DNA integrity was evaluated via agarose gel electrophoresis. To verify the suitability of the extracted DNA for downstream applications, we conducted 16S rRNA and ITS PCR amplifications.

Our findings demonstrated that our DNA extraction method produced significantly higher yields, better purity, and greater integrity compared to the commercial kit. Moreover, the extracted DNA was readily applicable in PCR-based procedures, confirming the method's effectiveness for molecular biology applications.


Palabras clave


DNA extraction; Chaotropic salt; Silica; PCR.

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Referencias


Alonso, A. (2013). DNA Extraction and Quantification. In Encyclopedia of Forensic Sciences: Second Edition (pp. 214–218). Elsevier Inc. https://doi.org/10.1016/B978-0-12-382165-2.00039-8

Becker, L., Steglich, M., Fuchs, S., Werner, G., & Nübel, U. (2016). Comparison of six commercial kits to extract bacterial chromosome and plasmid DNA for MiSeq sequencing. Scientific Reports, 6(June), 10–14. https://doi.org/10.1038/srep28063

Bernhardt, H. S., & Tate, W. P. (2012). Primordial soup or vinaigrette: Did the RNA world evolve at acidic pH? Biology Direct, 7(1), 4. https://doi.org/10.1186/1745-6150-7-4

Breeding, P., Wissenschafts-verlag, B., Issn, B., Biology, P., December, R., & February, A. (2004). Short Communication A new DNA extraction method for high-throughput marker analysis in a large-genome species such as Triticum aestivum. Matrix, 356(2001), 354–356.

Breeding, P., Wissenschafts-verlag, B., Issn, B., Biology, P., December, R., February, A., Sajali, N., Wong, S. C., Hanapi, U. K., Abu Bakar @ Jamaluddin, S., Tasrip, N. A., Mohd Desa, M. N., Demeke, T., Jenkins, G. R., Gupta, N., Singh, U. A., Kumari, M., Iyengar, S., Vilanova, S., … Prohens, J. (2018). Method for improving the quality of genomic DNA obtained from minute quantities of tissue and blood samples using Chelex 100 resin. Biological Procedures Online, 83(1), 1–11. https://doi.org/10.1186/s12575-018-0077-6

Chapela, M. J., Sotelo, C. G., Pérez-Martín, R. I., Pardo, M. Á., Pérez-Villareal, B., Gilardi, P., & Riese, J. (2007). Comparison of DNA extraction methods from muscle of canned tuna for species identification. Food Control, 18(10), 1211–1215. https://doi.org/10.1016/j.foodcont.2006.07.016

Dik, D. A., Zhang, N., Sturgell, E. J., Sanchez, B. B., Chen, J. S., Webb, B., Vanderpool, K. G., & Schultz, P. G. (2021). A synthetic 5,3-cross-link in the cell wall of rod-shaped Gram-positive bacteria. 118, 2100137118. https://doi.org/10.1073/pnas.2100137118/-/DCSupplemental

Dinis, T. B. V., Sousa, F., & Freire, M. G. (2020). Insights on the DNA Stability in Aqueous Solutions of Ionic Liquids. Frontiers in Bioengineering and Biotechnology, 8(October). https://doi.org/10.3389/fbioe.2020.547857

Dowd, C. J., & Kelley, B. (2011). Purification Process Design and the Influence of Product and Technology Platforms. In Comprehensive Biotechnology, Second Edition (Vol. 2, pp. 799–810). Elsevier Inc. https://doi.org/10.1016/B978-0-08-088504-9.00137-9

Earl, C. C., Smith, M. T., Lease, R. A., & Bundy, B. C. (2018). Polyvinylsulfonic acid: A low-cost rnase inhibitor for enhanced RNA preservation and cell-free protein translation. Bioengineered, 9(1), 90–97. https://doi.org/10.1080/21655979.2017.1313648

Garcia-Rubio, R., de Oliveira, H. C., Rivera, J., & Trevijano-Contador, N. (2020). The Fungal Cell Wall: Candida, Cryptococcus, and Aspergillus Species. In Frontiers in Microbiology (Vol. 10). Frontiers Media S.A. https://doi.org/10.3389/fmicb.2019.02993

Gontang, E. A., Fenical, W., & Jensen, P. R. (2007). Phylogenetic diversity of gram-positive bacteria cultured from marine sediments. Applied and Environmental Microbiology, 73(10), 3272–3282. https://doi.org/10.1128/AEM.02811-06

Goud, T. S., Upadhyay, R. C., Kumar, A., Karri, S., Choudhary, R., Ashraf, S., Singh, S. V., Kumar, O. S., & Kiranmai, C. (2018). Novel extraction of high quality genomic DNA from frozen bovine blood samples by using detergent method. Open Veterinary Journal, 8(4), 415–422. https://doi.org/10.4314/ovj.v8i4.11

Green, M. R., & Sambrook, J. (2017). Precipitation of DNA with isopropanol. Cold Spring Harbor Protocols, 2017(8), 673–674. https://doi.org/10.1101/pdb.prot093385

Günerken, E., D’Hondt, E., Eppink, M. H. M., Garcia-Gonzalez, L., Elst, K., & Wijffels, R. H. (2015). Cell disruption for microalgae biorefineries. Biotechnology Advances, 33(2), 243–260. https://doi.org/10.1016/j.biotechadv.2015.01.008

Gupta, N. (2019). DNA extraction and polymerase chain reaction. Journal of Cytology, 36(2), 116–117. https://doi.org/10.4103/JOC.JOC_110_18

Harrison, S. T. L. (2011). Cell Disruption. In Comprehensive Biotechnology, Second Edition (Vol. 2, pp. 619–640). Elsevier Inc. https://doi.org/10.1016/B978-0-08-088504-9.00127-6

Hosomi, K., Ohno, H., Murakami, H., Natsume-Kitatani, Y., Tanisawa, K., Hirata, S., Suzuki, H., Nagatake, T., Nishino, T., Mizuguchi, K., Miyachi, M., & Kunisawa, J. (2017). Method for preparing DNA from feces in guanidine thiocyanate solution affects 16S rRNA-based profiling of human microbiota diversity. Scientific Reports, 7(1), 1–10. https://doi.org/10.1038/s41598-017-04511-0

Hoyos, M., Tusso, S., Bedoya, T. R., Manrique Gaviria, A. S., & Bloor, P. (2017). A simple and cost-effective method for obtaining DNA from a wide range of animal wildlife samples. Conservation Genetics Resources, 9(4), 513–521. https://doi.org/10.1007/s12686-017-0735-z

Husakova, M., Kralik, P., Babak, V., & Slana, I. (2020). Efficiency of DNA isolation methods based on silica columns and magnetic separation tested for the detection of mycobacterium avium subsp. Paratuberculosis in milk and faeces. Materials, 13(22), 1–11. https://doi.org/10.3390/ma13225112

Islam, M. S., Aryasomayajula, A., & Selvaganapathy, P. R. (2017). A review on macroscale and microscale cell lysis methods. Micromachines, 8(3). https://doi.org/10.3390/mi8030083

J Shetty, P. (2020). The Evolution of DNA Extraction Methods. American Journal of Biomedical Science & Research, 8(1), 39–45. https://doi.org/10.34297/ajbsr.2020.08.001234

Jacobsen, N., Nielsen, P. S., Jeffares, D. C., Eriksen, J., Ohlsson, H., Arctander, P., Kauppinen, S., Hosomi, K., Ohno, H., Murakami, H., Natsume-Kitatani, Y., Tanisawa, K., Hirata, S., Suzuki, H., Nagatake, T., Nishino, T., Mizuguchi, K., Miyachi, M., Kunisawa, J., … Metzker, M. L. (2009). DNA, RNA, and protein extraction: The past and the present. Nucleic Acids Research, 4(2), 2440–2443. https://doi.org/10.1093/nar/gnh056

Jahanshahi, M., & Najafpour, G. (2007). Advanced Downstream Processing in Biotechnology. In Biochemical Engineering and Biotechnology (pp. 390–415). Elsevier. https://doi.org/10.1016/b978-044452845-2/50017-3

Jariwalla, R. J., Grossberg, S. E., & Sedmak, J. J. (1977). Effect of chaotropic salts and protein denaturants on the thermal stability of mouse fibroblast interferon. Journal of General Virology, 35(1), 45–52. https://doi.org/10.1099/0022-1317-35-1-45

K, K. (1999). Measurement of the rate of RNA hydrolysis in aqueous solution at elevated temperatures using a new monitoring method for hydrothermal reactions. Nucleic Acids Symposium Series, 42, 289–290. https://doi.org/10.1093/NASS/42.1.289

Katevatis, C., Fan, A., & Klapperich, C. M. (2017). Low concentration DNA extraction and recovery using a silica solid phase. PLoS ONE, 12(5), 1–14. https://doi.org/10.1371/journal.pone.0176848

Kumar, J., Kumar, M., Gupta, S., Ahmed, V., Bhambi, M., Pandey, R., & Chauhan, N. S. (2016). An Improved Methodology to Overcome Key Issues in Human Fecal Metagenomic DNA Extraction. Genomics, Proteomics and Bioinformatics, 14(6), 371–378. https://doi.org/10.1016/j.gpb.2016.06.002

Kumar, M., & Mugunthan, M. (2018). Evaluation of three DNA extraction methods from fungal cultures. Medical Journal Armed Forces India, 74(4), 333–336. https://doi.org/10.1016/j.mjafi.2017.07.009

Lemire, K. A., Rodriguez, Y. Y., & McIntosh, M. T. (2016). Alkaline hydrolysis to remove potentially infectious viral RNA contaminants from DNA. Virology Journal, 13(1). https://doi.org/10.1186/s12985-016-0552-0

Loparev, V. N., Cartas, M. A., Monken, C. E., Velpandi, A., & Srinivasan, A. (1991). An efficient and simple method of DNA extraction from whole blood and cell lines to identify infectious agents. In Journal of Virological Methods (Vol. 34).

Ma, F., Liu, F., Xu, W., & Li, L. (2018). Surfactant and Chaotropic Agent Assisted Sequential Extraction/On-Pellet Digestion (SCAD) for Enhanced Proteomics. Journal of Proteome Research, 17(8), 2744–2754. https://doi.org/10.1021/acs.jproteome.8b00197

Ma, J., Su, C., Hu, S., Chen, Y., Shu, Y., Yue, D., Zhang, B., Qi, Z., Li, S., Wang, X., Kuang, Y., & Cheng, P. (2020). The Effect of Residual Triton X-100 on Structural Stability and Infection Activity of Adenovirus Particles. Molecular Therapy - Methods and Clinical Development, 19, 35–46. https://doi.org/10.1016/j.omtm.2020.08.013

Mäki, A., Salmi, P., Mikkonen, A., Kremp, A., & Tiirola, M. (2017). Sample Preservation, DNA or RNA extraction and data analysis for high-throughput phytoplankton community sequencing. Frontiers in Microbiology, 8(SEP), 1–13. https://doi.org/10.3389/fmicb.2017.01848

Menezes, J. P., Lupatini, M., Antoniolli, Z. I., Blume, E., Junges, E., & Manzoni, C. G. (2010). Variabilidade genética na região its do rDNA de isolados de trichoderma spp. (Biocontrolador) e Fusarium oxysporum f. sp. Chrysanthemi. Ciência e Agrotecnologia, 34(1), 132–139. https://doi.org/10.1590/s1413-70542010000100017

Nouws, S., Bogaerts, B., Verhaegen, B., Denayer, S., Piérard, D., Marchal, K., Roosens, N. H. C., Vanneste, K., & De Keersmaecker, S. C. J. (2020). Impact of DNA extraction on whole genome sequencing analysis for characterization and relatedness of Shiga toxin-producing Escherichia coli isolates. Scientific Reports, 10(1), 1–16. https://doi.org/10.1038/s41598-020-71207-3

Peach, M., Marsh, N., Miskiewicz, E. I., & MacPhee, D. J. (2015). Solubilization of proteins: The importance of lysis buffer choice. In Western Blotting: Methods and Protocols (pp. 49–60). Springer New York. https://doi.org/10.1007/978-1-4939-2694-7_8

Pouseele, H., & Supply, P. (2015). Accurate whole-genome sequencing-based epidemiological surveillance of mycobacterium tuberculosis. In Methods in Microbiology (1st ed., Vol. 42). Elsevier Ltd. https://doi.org/10.1016/bs.mim.2015.04.001

Ren, X., Yu, D., Yu, L., Gao, G., Han, S., & Feng, Y. (2007). A new study of cell disruption to release recombinant thermostable enzyme from Escherichia coli by thermolysis. Journal of Biotechnology, 129(4), 668–673. https://doi.org/10.1016/j.jbiotec.2007.01.038

Sajali, N., Wong, S. C., Hanapi, U. K., Abu Bakar @ Jamaluddin, S., Tasrip, N. A., & Mohd Desa, M. N. (2018). The Challenges of DNA Extraction in Different Assorted Food Matrices: A Review. Journal of Food Science, 83(10), 2409–2414. https://doi.org/10.1111/1750-3841.14338

Salimans, M., & Sol, C. J. A. (1990). Rapid and Simple Method for Purification of Nucleic Acids. 495–503.

Salvi, G., De Los Rios, P., & Vendruscolo, M. (2005). Effective interactions between chaotropic agents and proteins. Proteins: Structure, Function and Genetics, 61(3), 492–499. https://doi.org/10.1002/prot.20626

Sawyer, W. H., & Puckridge, J. (1973). The dissociation of proteins by chaotropic salts. Journal of Biological Chemistry, 248(24), 8429–8433. https://doi.org/10.1016/s0021-9258(19)43151-7

Scorsato, A. P., & Telles, J. E. Q. (2011). Factors that affect the quality of DNA extracted from biological samples stored in paraffin blocks. Jornal Brasileiro de Patologia e Medicina Laboratorial, 47(5), 541–548. https://doi.org/10.1590/s1676-24442011000500008

Sedlackova, T., Repiska, G., Celec, P., Szemes, T., & Minarik, G. (2013). Fragmentation of DNA affects the accuracy of the DNA quantitation by the commonly used methods. Biological Procedures Online, 15(1). https://doi.org/10.1186/1480-9222-15-5

Shamsi, M. B., Imam, S. N., & Dada, R. (2011). Sperm DNA integrity assays: Diagnostic and prognostic challenges and implications in management of infertility. Journal of Assisted Reproduction and Genetics, 28(11), 1073–1085. https://doi.org/10.1007/s10815-011-9631-8

Verma, P. V., Vasudevan, V., Kashyap, B. K., Samsudeen, T. I., Meghvansi, M. K., Kamboj, D. V., & Singh, L. (2018). Direct Lysis Glass Milk Method of Genomic Dna Extraction Reveals Greater Archaeal Diversity in Anaerobic Biodigester Slurry As Assessed Through Denaturing Gradient Gel Electrophoresis. Journal of Experimental Biology and Agricultural Sciences, 6(2), 315–323. https://doi.org/10.18006/2018.6(2).315.323

Verma, S. K., Singh, H., & Sharma, P. C. (2017). An improved method suitable for isolation of high-quality metagenomic DNA from diverse soils. 3 Biotech, 7(3), 1–7. https://doi.org/10.1007/s13205-017-0847-x

Wang, W., Wang, Y. J., & Wang, D. Q. (2008). Dual effects of Tween 80 on protein stability. International Journal of Pharmaceutics, 347(1–2), 31–38. https://doi.org/10.1016/j.ijpharm.2007.06.042

Wohlgemuth, R. (2019). Product recovery. In Comprehensive Biotechnology (pp. 681–691). Elsevier. https://doi.org/10.1016/B978-0-444-64046-8.00096-3

Zainabadi, K., Dhayabaran, V., Moideen, K., & Krishnaswamy, P. (2019). An efficient and cost-effective method for purification of small sized DNAs and RNAs from human urine. PLoS ONE, 14(2), 1–15. https://doi.org/10.1371/journal.pone.0210813




DOI: https://doi.org/10.23857/pc.v9i10.8152

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