Isolation of fungal genomic DNA of high quality is required for a number of downstream biotechnology-derived applications such as genome sequencing, microarrays, and digital PCR technologies, to only name a few. In most cases, not only a high molecular weight DNA of superior grade is required but also large quantities. On the other hand, a number of laboratory experiments, such as polymerase chain reaction (PCR) for medical diagnostic or for genotyping, have to be conducted in a limited amount of time and can provide complete results with the use of lower quality DNA. We describe here two different fungal DNA extraction approaches, which are applicable to a wide range of fungal species.First, we adapted a DNA extraction method for PCR-based genotyping which allows analysis of single to hundreds of colonies simultaneously. Cells are disrupted in the presence of sodium dodecyl sulfate and Proteinase K which are then removed by precipitation and centrifugation. The cleared lysate is used for PCR reaction.Secondly, we describe a method to obtain genome sequencing quality grade DNA from fungal liquid cultures. Mycelia are harvested by either filtration or centrifugation. Cells are mechanically disrupted by liquid nitrogen grinding, followed by genomic DNA extraction using the QIAGEN's DNeasy ® Plant Kit. The quality and quantity of genomic DNA is monitored by fluorometry.
Keywords: Agarose gel electrophoresis; Colony PCR; DNA quantification; Fluorometry; Fungal genomic DNA extraction; Mycelia; Spectrophotometer.
Serna-Domínguez MG, Andrade-Michel GY, Arredondo-Bernal HC, Gallou A. Serna-Domínguez MG, et al. J Microbiol Methods. 2018 May;148:55-63. doi: 10.1016/j.mimet.2018.03.012. Epub 2018 Mar 27. J Microbiol Methods. 2018. PMID: 29596959
Zhang YJ, Zhang S, Liu XZ, Wen HA, Wang M. Zhang YJ, et al. Lett Appl Microbiol. 2010 Jul;51(1):114-8. doi: 10.1111/j.1472-765X.2010.02867.x. Epub 2010 May 14. Lett Appl Microbiol. 2010. PMID: 20536704
Manian S, Sreenivasaprasad S, Mills PR. Manian S, et al. Lett Appl Microbiol. 2001 Oct;33(4):307-10. doi: 10.1046/j.1472-765x.2001.01001.x. Lett Appl Microbiol. 2001. PMID: 11559406
Atkins SD, Clark IM. Atkins SD, et al. J Appl Genet. 2004;45(1):3-15. J Appl Genet. 2004. PMID: 14960763 Review.
Sharma KK. Sharma KK. Crit Rev Biotechnol. 2016 Aug;36(4):743-59. doi: 10.3109/07388551.2015.1015959. Epub 2015 Feb 27. Crit Rev Biotechnol. 2016. PMID: 25721271 Review.
Pham D, Sivalingam V, Tang HM, Montgomery JM, Chen SC, Halliday CL. Pham D, et al. J Fungi (Basel). 2024 Jun 26;10(7):447. doi: 10.3390/jof10070447. J Fungi (Basel). 2024. PMID: 39057332 Free PMC article. Review.
Ramos MLM, Almeida-Silva F, de Souza Rabello VB, Nahal J, Figueiredo-Carvalho MHG, Bernardes-Engemann AR, Poester VR, Xavier MO, Meyer W, Zancopé-Oliveira RM, Frases S, Almeida-Paes R. Ramos MLM, et al. Braz J Microbiol. 2024 Jun;55(2):1359-1368. doi: 10.1007/s42770-024-01301-5. Epub 2024 Mar 11. Braz J Microbiol. 2024. PMID: 38466550
Mathieu Y, Raji O, Bellemare A, Di Falco M, Nguyen TTM, Viborg AH, Tsang A, Master E, Brumer H. Mathieu Y, et al. Biotechnol Biofuels Bioprod. 2023 Sep 7;16(1):132. doi: 10.1186/s13068-023-02383-3. Biotechnol Biofuels Bioprod. 2023. PMID: 37679837 Free PMC article.