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A Quick and Safe Method for Fungal DNA Extraction

  • Chi, Myoung-Hwan (Department of Agricultural Biotechnology, Center for Fungal Genetic Resources, and Center for Fungal Pathogenesis, Seoul National University) ;
  • Park, Sook-Young (Department of Agricultural Biotechnology, Center for Fungal Genetic Resources, and Center for Fungal Pathogenesis, Seoul National University) ;
  • Lee, Yong-Hwan (Department of Agricultural Biotechnology, Center for Fungal Genetic Resources, and Center for Fungal Pathogenesis, Seoul National University)
  • Published : 2009.03.31

Abstract

DNA-based studies, including cloning and genotyping, have become routine in fungal research laboratories. However, preparation of high-quality DNA from fungal tissue requires much time and labor and is often a limiting step for high-throughput experiments. We have developed a quick and safe (QS) DNA extraction method for fungi. Time efficiency and safety in the QS method were achieved by using plate-grown mycelia as the starting material, by eliminating phenol-chloroform extraction procedures, and by deploying a simple electric grinder. This QS method is applicable not only to a broad range of microbial eukaryotes, including true fungi and oomycetes, but also to lichens and plants.

Keywords

fungi;genomic DNA extraction;high-throughput

References

  1. Loeffler, J., Schmidt, K., Hebart, B., Schumachcr, U. and Einsclc, H. 2002. Automated extraction of genomic DNA from medically important yeast species and filamentous fungi by using the MagNA Pure LC system. J Clin. Microbial. 40:2240-3 https://doi.org/10.1128/JCM.40.6.2240-2243.2002
  2. Mulier, F. M. C., Wemer. K. E., Kasai, Moo Francesconi, A., Chanock, S. J. and Walsh, T. J. 1998. Rapid extraction of genomic DNA from medically important yeasts and filamentous fungi by high-speed cell disruption. J. Clin. Microbial. 36: 1625-1629
  3. Thomson, D. and Henry, R. 1995. Single-step protocol for preparation of plant-tissue for analysis by PCR. Biotechniques 19:394-400
  4. Liu. D., Coloe. S., Baird, R. and Pedersen, J. 2000. Rapid minipreparation of fungal DNA for PCR. J Clin. Microhiol. 38:471
  5. Cenis, J. L. 1992. Rapid extraction of fungal DNA for PCR amplitication. Nucleic Acids Res. 20:2380 https://doi.org/10.1093/nar/20.9.2380
  6. Lugert, R., Schettler, C. and Gross, U. 2006. Comparison of different protocols for DNA preparation and PCR for the detection offungal pathogens in vitro. Mycoses 49:298-304 https://doi.org/10.1111/j.1439-0507.2006.01255.x
  7. Guo, J. R .. Schnieder, E, Abd-Elsalam, K. A. and Verreet, J. A. 2005. Rapid and efficient extraction of gGuo, J. R .. Schnieder, E, Abd-Elsalam, K. A. and Verreet, J. A.enomic DNA from ditlerent phytopathogenic fungi using DNAzol reagent. Bioteelmol. Lett. 27:3-6
  8. Borman, A. M., Linton, C. J., Miles, S. J., Campbell, C. K. and Johnson, E. M. 2006. Ultra-rapid preparation of total genomic DNA from isolates of yeast and mould using Whatman FTA filter paper technology - a reusable DNA archiving system. Med Mycol. 44:389-398 https://doi.org/10.1080/13693780600564613
  9. Cassago. A., Panepucci, R. A., Baiao, A. and Henrique-Silva, F. 2002. Cellophane based mini-prep method for DNA extraction from the filamentous fungus Trichoderma reesei. BMC Microhiol. 2: 14 https://doi.org/10.1186/1471-2180-2-14
  10. Rogers, S. O. and Bendich, A. J. 1985. Extraction of DNA from milligram amounts of fresh, herbarium and mummitied planttissues. Plant Mol. Biol. 5:69-76 https://doi.org/10.1007/BF00020088

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  2. A quick and accurate screening method for fungal gene-deletion mutants by direct, priority-based, and inverse PCRs vol.105, 2014, https://doi.org/10.1016/j.mimet.2014.06.023
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  4. Systematic characterization of the peroxidase gene family provides new insights into fungal pathogenicity in Magnaporthe oryzae vol.5, pp.1, 2015, https://doi.org/10.1038/srep11831
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  9. Genome-wide analyses of DNA-binding proteins harboring AT-hook motifs and their functional roles in the rice blast pathogen, Magnaporthe oryzae vol.36, pp.6, 2014, https://doi.org/10.1007/s13258-014-0233-6
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  18. Chitin-deacetylase activity induces appressorium differentiation in the rice blast fungus Magnaporthe oryzae vol.7, pp.1, 2017, https://doi.org/10.1038/s41598-017-10322-0
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  23. A Novel Pathogenicity Gene Is Required in the Rice Blast Fungus to Suppress the Basal Defenses of the Host vol.5, pp.4, 2009, https://doi.org/10.1371/journal.ppat.1000401
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  25. Characterization of Monilinia fructicola Strains Resistant to Both Propiconazole and Boscalid vol.97, pp.5, 2013, https://doi.org/10.1094/PDIS-10-12-0924-RE
  26. Specific PCR Detection of Four Quarantine Fusarium Species in Korea vol.26, pp.4, 2010, https://doi.org/10.5423/PPJ.2010.26.4.409
  27. Structure and function of the mating-type locus in the homothallic ascomycete, Didymella zeae-maydis vol.51, pp.6, 2013, https://doi.org/10.1007/s12275-013-3465-2
  28. Field Strains of Monilinia fructicola Resistant to Both MBC and DMI Fungicides Isolated from Stone Fruit Orchards in the Eastern United States vol.97, pp.8, 2013, https://doi.org/10.1094/PDIS-12-12-1177-RE
  29. M233I Mutation in the β-Tubulin of Botrytis cinerea Confers Resistance to Zoxamide vol.5, pp.1, 2015, https://doi.org/10.1038/srep16881
  30. Multiple Fungicide Resistance in Botrytis cinerea from Greenhouse Strawberries in Hubei Province, China vol.101, pp.4, 2017, https://doi.org/10.1094/PDIS-09-16-1227-RE
  31. Location-Specific Fungicide Resistance Profiles and Evidence for Stepwise Accumulation of Resistance in Botrytis cinerea vol.98, pp.8, 2014, https://doi.org/10.1094/PDIS-10-13-1019-RE
  32. Characterization of Resistance to Six Chemical Classes of Site-Specific Fungicides Registered for Gray Mold Control on Strawberry in Spain vol.100, pp.11, 2016, https://doi.org/10.1094/PDIS-03-16-0280-RE
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  34. Fitness and Competitive Ability of Botrytis cinerea Isolates with Resistance to Multiple Chemical Classes of Fungicides vol.106, pp.9, 2016, https://doi.org/10.1094/PHYTO-02-16-0061-R
  35. Relationship between expression level of hygromycin B-resistant gene and Agrobacterium tumefaciens -mediated transformation efficiency in Beauveria bassiana JEF-007 vol.123, pp.3, 2017, https://doi.org/10.1111/jam.13529
  36. Production of antibacterial Bombyx mori cecropin A in mealworm-pathogenic Beauveria bassiana ERL1170 vol.42, pp.1, 2015, https://doi.org/10.1007/s10295-014-1551-z
  37. Systematic Analysis of the Anticancer Agent Taxol-Producing Capacity inColletotrichumSpecies and Use of the Species for Taxol Production vol.44, pp.2, 2016, https://doi.org/10.5941/MYCO.2016.44.2.105
  38. Resistance to Pyraclostrobin and Boscalid in Botrytis cinerea Isolates from Strawberry Fields in the Carolinas vol.96, pp.8, 2012, https://doi.org/10.1094/PDIS-12-11-1049-RE
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  45. The cell cycle gene MoCDC15 regulates hyphal growth, asexual development and plant infection in the rice blast pathogen Magnaporthe oryzae vol.48, pp.8, 2011, https://doi.org/10.1016/j.fgb.2011.05.001
  46. Genome-wide profiling of DNA methylation provides insights into epigenetic regulation of fungal development in a plant pathogenic fungus, Magnaporthe oryzae vol.5, pp.1, 2015, https://doi.org/10.1038/srep08567
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  49. Trichoderma Biodiversity of Agricultural Fields in East China Reveals a Gradient Distribution of Species vol.11, pp.8, 2016, https://doi.org/10.1371/journal.pone.0160613
  50. A split luciferase complementation assay for studying in vivo protein–protein interactions in filamentous ascomycetes vol.58, pp.3, 2012, https://doi.org/10.1007/s00294-012-0375-5
  51. Expression of egfp gene based on Agrobacterium tumefaciens-mediated transformation in Beauveria bassiana sensu lato ERL836 vol.18, pp.4, 2015, https://doi.org/10.1016/j.aspen.2015.07.013
  52. Multiple roles of a putative vacuolar protein sorting associated protein 74, FgVPS74, in the cereal pathogen Fusarium graminearum vol.53, pp.4, 2015, https://doi.org/10.1007/s12275-015-5067-7
  53. Characterization of Nivalenol-Producing Fusarium culmorum Isolates Obtained from the Air at a Rice Paddy Field in Korea vol.32, pp.3, 2016, https://doi.org/10.5423/PPJ.OA.12.2015.0268
  54. Role of MoAND1-mediated nuclear positioning in morphogenesis and pathogenicity in the rice blast fungus, Magnaporthe oryzae vol.69, 2014, https://doi.org/10.1016/j.fgb.2014.05.002
  55. Effect of Fungicide Applications on Monilinia fructicola Population Diversity and Transposon Movement vol.106, pp.12, 2016, https://doi.org/10.1094/PHYTO-03-16-0127-R
  56. An Easy, Rapid, and Cost-Effective Method for DNA Extraction from Various Lichen Taxa and Specimens Suitable for Analysis of Fungal and Algal Strains vol.42, pp.4, 2014, https://doi.org/10.5941/MYCO.2014.42.4.311
  57. Functional Roles of FgLaeA in Controlling Secondary Metabolism, Sexual Development, and Virulence in Fusarium graminearum vol.8, pp.7, 2013, https://doi.org/10.1371/journal.pone.0068441
  58. Botrytis caroliniana, a new species isolated from blackberry in South Carolina vol.104, pp.3, 2012, https://doi.org/10.3852/11-218
  59. Resistance to the SDHI Fungicides Boscalid, Fluopyram, Fluxapyroxad, and Penthiopyrad in Botrytis cinerea from Commercial Strawberry Fields in Spain vol.101, pp.7, 2017, https://doi.org/10.1094/PDIS-01-17-0067-RE
  60. Two conidiation-related Zn(II)2Cys6 transcription factor genes in the rice blast fungus vol.61, 2013, https://doi.org/10.1016/j.fgb.2013.10.004
  61. Analysis of in planta Expressed Orphan Genes in the Rice Blast Fungus Magnaporthe oryzae vol.30, pp.4, 2014, https://doi.org/10.5423/PPJ.OA.08.2014.0072
  62. in Korea vol.102, pp.5, 2010, https://doi.org/10.3852/09-304
  63. Bidirectional-Genetics Platform, a Dual-Purpose Mutagenesis Strategy for Filamentous Fungi vol.12, pp.11, 2013, https://doi.org/10.1128/EC.00234-13
  64. Identification of Fusarium subglutinans, the Casual Pathogen of Corn Stalk Rot in Korea and Investigation of Effectiveness of Fungicides Against the Pathogen vol.48, pp.3, 2014, https://doi.org/10.14397/jals.2014.48.3.43
  65. in California Strawberry Fields pp.0191-2917, 2018, https://doi.org/10.1094/PDIS-03-18-0406-RE
  66. Distinct roles of the YPEL gene family in development and pathogenicity in the ascomycete fungus Magnaporthe oryzae vol.8, pp.1, 2018, https://doi.org/10.1038/s41598-018-32633-6
  67. vol.102, pp.8, 2018, https://doi.org/10.1094/PDIS-12-17-1933-RE