Fungal Genomic DNA Extraction Methods for Rapid Genotyping and Genome Sequencing

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.

Genomic DNA extraction from minimal amount of dried mushroom samples

“…A stronger concentration of SDS was used in the current study in the comparison to the previous reports (Bellemare et al, 2018;Müller et al, 1998;Umesha et al, 2016) in order to solubilise the proteins and lipids in the cell membranes, while simultaneously denaturing proteins in the cytoplasm at a faster rate. Expensive and toxic reagents were avoided, including liquid nitrogen and hydrogen chloride, which is required by some protocols (Avin et al, 2012;Lahuf et al, 2019;Yang et al, 2016).…”

Modified Phenol/Chloroform‐Free DNA Isolation from Yeast and Other Fungi by Non‐Enzymatic Salting Out Method

“…Isolation of genomic DNA from fungi is necessary for clinical diagnosis and biotechnological research (Bellemare et al., 2018; Tsui et al., 2011). In this study, we have proposed a rapid and safe procedure for extracting the fungal DNA genome based on the salting‐out method.…”

Screening of novel fungal Carbohydrate Esterase family 1 enzymes identifies three novel dual feruloyl/acetyl xylan esterases

“…Gene sequences were amplified by PCR using genomic DNA or cDNA as the template DNA. Genomic DNA was extracted using the DNeasyÒ Plant Mini kit (QIAGEN, Hilden, Germany) as previously described [39], and cDNA was obtained following the previously described method [40]. The genes were cloned using an in-house variation of the ligation-independent cloning (LIC) method [41].…”