Abstract
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.
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References
Fredricks DN, Smith C, Meier A (2005) Comparison of six DNA extraction methods for recovery of fungal DNA as assessed by quantitative PCR. J Clin Microbiol 43(10):5122–5128
Tan SC, Yiap BC (2009) DNA, RNA, and protein extraction: the past and the present. J Biomed Biotechnol 2009:574398
Alshahni MM et al (2009) Direct colony PCR of several medically important fungi using Ampdirect plus. Jpn J Infect Dis 62(2):164–167
Umesha S, Manukumar HM, Raghava S (2016) A rapid method for isolation of genomic DNA from food-borne fungal pathogens. 3 Biotech 6(2):123
Khot PD, Fredricks DN (2009) PCR-based diagnosis of human fungal infections. Expert Rev Anti-Infect Ther 7(10):1201–1221
Black JA, Foarde KK (2007) Comparison of four different methods for extraction of Stachybotrys chartarum spore DNA and verification by real-time PCR. J Microbiol Methods 70(1):75–81
Demeke T, Jenkins GR (2010) Influence of DNA extraction methods, PCR inhibitors and quantification methods on real-time PCR assay of biotechnology-derived traits. Anal Bioanal Chem 396(6):1977–1990
Liles MR et al (2008) Recovery, purification, and cloning of high-molecular-weight DNA from soil microorganisms. Appl Environ Microbiol 74(10):3302–3305
Robin JD et al (2016) Comparison of DNA quantification methods for next generation sequencing. Sci Rep 6:24067
Taskova RM et al (2006) A comparison of cell wall disruption techniques for the isolation of intracellular metabolites from Pleurotus and Lepista sp. Z Naturforsch C 61(5–6):347–350
Zachová I et al (2003) Detection of aflatoxigenic fungi in feeds using the PCR method. Folia Microbiol 48(6):817–821
Weyant RS, Edmonds P, Swaminathan B (1990) Effect of ionic and nonionic detergents on the Taq polymerase. BioTechniques 9(3):308–309
Acknowledgment
This work was supported by Genome Canada and Génome Québec.
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Bellemare, A., John, T., Marqueteau, S. (2018). Fungal Genomic DNA Extraction Methods for Rapid Genotyping and Genome Sequencing. In: de Vries, R., Tsang, A., Grigoriev, I. (eds) Fungal Genomics. Methods in Molecular Biology, vol 1775. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7804-5_2
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DOI: https://doi.org/10.1007/978-1-4939-7804-5_2
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Publisher Name: Humana Press, New York, NY
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Online ISBN: 978-1-4939-7804-5
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