DNA Extraction from Fungi: Protocols for Yeast and Mold

DNA extraction from fungi presents unique challenges due to the rigid cell walls that characterize both yeast and filamentous fungi. Unlike bacterial or mammalian cells, fungal cells require specialized lysis methods—enzymatic, mechanical, or chemical—to release high-quality genomic DNA suitable for downstream applications such as PCR, sequencing, and metagenomic analysis. This article provides evidence-based protocols for extracting DNA from yeast and mold, emphasizing method selection based on sample type, desired DNA yield and integrity, and intended application. The protocols described are appropriate for BSL-1 laboratory settings and exclude pathogenic or select-agent organisms.

At a Glance

Scientific Principle

Fungal DNA extraction relies on disrupting the cell wall and membrane to release nucleic acids, followed by purification to remove proteins, polysaccharides, and other contaminants. The fungal cell wall is a complex structure composed primarily of chitin, glucans, and mannoproteins, which varies significantly between yeast and filamentous fungi [1]. Yeast cell walls are typically thinner and more susceptible to enzymatic digestion, while filamentous fungi often require mechanical disruption to break hyphal structures.

The choice of lysis method directly impacts DNA quality and yield. Enzymatic lysis using lyticase or zymolyase specifically degrades β-1,3-glucan linkages in yeast cell walls, providing gentle lysis that preserves DNA integrity [1]. Mechanical lysis via bead beating physically shears cell walls but can also fragment DNA, making it less suitable for applications requiring high molecular weight DNA, such as long-read sequencing. Chemical lysis using cetyltrimethylammonium bromide (CTAB) or sodium dodecyl sulfate (SDS) solubilizes membranes and denatures proteins while protecting nucleic acids from nucleases [1].

After lysis, DNA is typically purified through organic extraction (phenol-chloroform) or solid-phase methods (silica columns, magnetic beads). The choice of purification method affects DNA purity, yield, and suitability for downstream applications. For metagenomic studies, the extraction method must efficiently lyse diverse fungal taxa while minimizing bias toward easily disrupted cells [3].

Materials and Instrumentation

Reagents

• Lysis enzymes: Lyticase (from Arthrobacter luteus) or zymolyase (from Streptomyces sp.) for yeast cell wall digestion. These enzymes are available as lyophilized powders and should be reconstituted in sterile water or buffer immediately before use.
• Mechanical disruption media: Acid-washed glass beads (0.5 mm diameter for yeast, 0.1–0.5 mm for filamentous fungi), zirconia/silica beads, or stainless steel beads. Bead size affects lysis efficiency: smaller beads provide more surface area but may generate more heat.
• Lysis buffers: CTAB extraction buffer (2% CTAB, 100 mM Tris-HCl pH 8.0, 20 mM EDTA, 1.4 M NaCl) for chemical lysis; SDS-based buffers (e.g., 2% SDS, 100 mM Tris-HCl pH 8.0, 50 mM EDTA) for alternative chemical approaches.
• Purification reagents: Phenol:chloroform:isoamyl alcohol (25:24:1), chloroform:isoamyl alcohol (24:1), isopropanol, ethanol (70% and 100%), sodium acetate (3 M, pH 5.2), or commercial DNA binding buffers for column-based purification.
• RNase: DNase-free RNase A (10 mg/mL) to remove RNA contamination.
• Wash buffers: For column-based methods, typically containing ethanol and chaotropic salts.

Equipment

• Homogenizer: Bead mill (e.g., TissueLyser, FastPrep, Mini-Beadbeater) for mechanical lysis. Vortex adapters can substitute for small sample numbers.
• Centrifuge: Refrigerated microcentrifuge capable of 12,000–16,000 × g for DNA precipitation and column steps.
• Incubator or heat block: For enzymatic digestion (30–37°C) and chemical lysis (60–65°C).
• Spectrophotometer: NanoDrop or similar for assessing DNA concentration and purity (260/280 and 260/230 ratios).
• Electrophoresis equipment: Agarose gel electrophoresis system for DNA integrity assessment.
• Water bath or thermal mixer: For controlled temperature incubations.

Consumables

• Sterile microcentrifuge tubes (1.5 mL, 2 mL)
• Sterile pipette tips with aerosol barriers
• Disposable gloves
• DNA-free water (molecular biology grade)

Controls

Appropriate controls are essential for validating DNA extraction efficiency and identifying contamination. Include the following:

• Positive extraction control: A known fungal strain (e.g., Saccharomyces cerevisiae for yeast, Aspergillus niger for mold) processed alongside samples to verify lysis and purification efficiency.
• Negative extraction control: Sterile water or buffer processed through the entire extraction procedure to detect reagent contamination.
• No-template control: For downstream PCR applications, include a water-only control to detect amplicon contamination.
• Spike-in control: For environmental or mixed samples, add a known quantity of exogenous DNA (e.g., from a non-target organism) to monitor recovery efficiency and inhibition in downstream assays.

Document all control results in laboratory notebooks, including lot numbers of reagents and dates of preparation.

Conceptual Workflow

Step 1: Sample Preparation

For pure yeast cultures: Harvest cells from liquid culture (typically 10⁷–10⁸ cells) by centrifugation at 3,000–5,000 × g for 5 minutes. Wash cells with sterile water or PBS to remove culture medium components that may inhibit downstream reactions. For solid media, scrape colonies and resuspend in lysis buffer.

For filamentous fungi: Harvest mycelia from liquid culture by filtration through sterile Miracloth or cheesecloth, or scrape from agar plates. Blot excess liquid with sterile filter paper. For environmental samples (e.g., soil, dust, book surfaces), process as described in [2] and [4], noting that desiccation-tolerant species may require modified lysis conditions.

For clinical specimens: Follow institutional biosafety protocols. For hemoculture specimens, human DNA depletion may be necessary to enrich fungal DNA [1].

Step 2: Cell Lysis

Enzymatic lysis (yeast): Resuspend cell pellet in 200–500 µL of sorbitol buffer (1 M sorbitol, 100 mM sodium citrate, 10 mM EDTA, pH 7.0) containing 1–5 U of lyticase or zymolyase. Incubate at 30°C for 30–60 minutes with gentle agitation. Monitor spheroplast formation by phase-contrast microscopy or by measuring OD₆₀₀ reduction after dilution in water. Spheroplasts are osmotically sensitive and should be handled gently.

Mechanical lysis (filamentous fungi): Transfer mycelia to a 2 mL tube containing 0.5–1 g of acid-washed glass beads and 500 µL of lysis buffer. Homogenize in a bead mill at maximum speed for 30–60 seconds. Repeat 2–3 times with 1-minute cooling intervals on ice to prevent DNA shearing from heat generation. For tough hyphae, increase bead-beating time or use zirconia/silica beads.

Chemical lysis (both yeast and mold): Add 500 µL of CTAB extraction buffer preheated to 65°C. Incubate at 65°C for 30–60 minutes with occasional inversion. CTAB complexes with polysaccharides and removes them during organic extraction [1]. For SDS-based lysis, incubate at 60°C for 30 minutes.

Step 3: Purification

Organic extraction: Add an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1). Vortex thoroughly and centrifuge at 12,000 × g for 10 minutes at 4°C. Transfer the aqueous (upper) phase to a new tube. Repeat with chloroform:isoamyl alcohol (24:1) to remove residual phenol. Precipitate DNA by adding 0.1 volumes of 3 M sodium acetate (pH 5.2) and 2.5 volumes of ice-cold 100% ethanol. Incubate at -20°C for at least 30 minutes (or overnight for maximum yield). Centrifuge at 12,000 × g for 15 minutes at 4°C. Wash pellet with 70% ethanol, air-dry, and resuspend in 50–100 µL of DNA-free water or TE buffer.

Column-based purification: Follow manufacturer instructions for the specific kit. Typically, bind DNA to silica membrane in the presence of chaotropic salts, wash with ethanol-containing buffers, and elute in low-ionic-strength buffer. Column methods are faster and avoid hazardous organic solvents but may yield lower DNA amounts for some fungal species.

Step 4: Quality Assessment

Assess DNA concentration and purity using spectrophotometry. A 260/280 ratio of 1.8–2.0 indicates minimal protein contamination; ratios below 1.8 suggest protein or phenol carryover. A 260/230 ratio of 2.0–2.2 indicates low polysaccharide or guanidine contamination; lower ratios suggest residual contaminants that may inhibit downstream applications [1].

Assess DNA integrity by agarose gel electrophoresis (0.8–1% gel). High molecular weight DNA appears as a single band >10 kb with minimal smearing. For long-read sequencing applications, DNA fragments should exceed 10 kb [1]. For PCR-based applications, some fragmentation is acceptable.

Quality Checks

• Spectrophotometric ratios: Record 260/280 and 260/230 for each sample. Re-purify samples with ratios outside acceptable ranges.
• Gel electrophoresis: Visualize DNA on agarose gel. Note any smearing (indicating degradation) or high molecular weight bands (indicating intact DNA).
• Quantification: Use fluorometric methods (e.g., Qubit) for accurate quantification, especially for low-concentration samples or those containing contaminants that absorb at 260 nm.
• PCR amplification: Test DNA quality by amplifying a single-copy fungal gene (e.g., ITS region, β-tubulin). Failed amplification may indicate inhibitors or degraded DNA.
• Negative control verification: Confirm no visible DNA or PCR product in negative extraction controls.

Result Interpretation

Successful DNA extraction yields sufficient, pure, and intact DNA for the intended application. For PCR-based methods, 1–50 ng of DNA per reaction is typically sufficient. For long-read sequencing, 1–5 µg of high molecular weight DNA (>10 kb) is required [1].

Low 260/280 ratios (<1.8) indicate protein contamination, which can be addressed by additional phenol:chloroform extraction or proteinase K treatment. Low 260/230 ratios (<2.0) suggest polysaccharide or chaotropic salt contamination; for column-purified DNA, additional wash steps may help. For CTAB-extracted DNA, residual CTAB can cause low 260/230 ratios and inhibit PCR; additional ethanol precipitation or column cleanup may be necessary.

DNA degradation (smearing on gel) may result from excessive mechanical shearing, nuclease activity, or improper storage. To minimize degradation, keep samples on ice during processing, use nuclease-free reagents, and store DNA at -20°C or -80°C for long-term storage.

Troubleshooting

Limitations

DNA extraction from fungi has several inherent limitations that researchers should consider:

• Cell wall variability: Different fungal species have vastly different cell wall compositions. A protocol optimized for Saccharomyces cerevisiae may not efficiently lyse Aspergillus fumigatus or xerophilic species like Aspergillus halophilicus [2]. Pilot experiments with target organisms are essential.
• DNA shearing: Mechanical lysis methods, while effective for tough cell walls, can fragment DNA, limiting suitability for long-read sequencing. Enzymatic methods preserve DNA integrity but may not lyse all species [1].
• Polysaccharide contamination: Many fungi produce polysaccharides that co-precipitate with DNA and inhibit downstream enzymes. CTAB extraction helps remove polysaccharides but may not eliminate all contaminants.
• Low biomass samples: Environmental samples (e.g., air filters, dust, book surfaces) may contain very low fungal biomass, requiring concentration steps or increased sample input [2, 4].
• Bias in mixed communities: No single extraction method efficiently lyses all fungal taxa equally. Metagenomic studies may underrepresent species with tough cell walls or overrepresent easily lysed species [3].
• Inhibitor carryover: Clinical and environmental samples often contain PCR inhibitors (e.g., heme, humic acids, melanin) that co-purify with DNA. Additional cleanup steps may be necessary [5].

Documentation

Maintain detailed records of all DNA extraction procedures, including:

• Sample source, collection date, and storage conditions
• Extraction method and any modifications
• Reagent lot numbers and expiration dates
• Incubation times and temperatures
• Centrifugation speeds and durations
• Quality control results (spectrophotometry, gel images, quantification)
• Storage location and conditions of extracted DNA

For clinical or environmental studies, document any deviations from standard protocols and their rationale. This documentation supports reproducibility and troubleshooting.

Biosafety Considerations

All work with fungal cultures should be performed in accordance with institutional biosafety guidelines and the principles outlined in the CDC/NIH Biosafety in Microbiological and Biomedical Laboratories (BMBL), 6th Edition [6]. For BSL-1 organisms (e.g., Saccharomyces cerevisiae, non-pathogenic Aspergillus species), standard microbiological practices apply:

• Perform all procedures in a designated laboratory area
• Use personal protective equipment (lab coat, gloves, safety glasses)
• Decontaminate work surfaces before and after procedures
• Dispose of biological waste according to institutional guidelines
• Avoid generating aerosols during bead beating; use sealed tubes

For unknown environmental isolates or samples from clinical settings, consult institutional biosafety officers to determine appropriate containment levels. Do not propagate or culture known pathogens without appropriate BSL-2 or higher facilities and training [6].

For work involving recombinant or synthetic nucleic acid molecules, follow the NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules [7]. Institutional biosafety committees must approve any experiments involving recombinant DNA.

Frequently Asked Questions

1. Can I use the same protocol for both yeast and filamentous fungi? No, the optimal protocol differs. Yeast cells are efficiently lysed by enzymatic digestion (lyticase/zymolyase), which preserves DNA integrity. Filamentous fungi typically require mechanical disruption (bead beating) to break hyphal cell walls. Chemical lysis with CTAB can work for both but may require optimization of incubation time and temperature. Always pilot-test protocols with your specific organism.

2. How do I choose between enzymatic and mechanical lysis for my application? Consider your downstream application. For long-read sequencing (e.g., Oxford Nanopore, PacBio), enzymatic lysis is preferred because it yields high molecular weight DNA (>10 kb) [1]. For PCR-based applications or short-read sequencing, mechanical lysis is acceptable and may be more efficient for tough cell walls. For metagenomic studies, use a method that balances lysis efficiency across diverse taxa [3].

3. Why is my DNA yield low even though I used a commercial kit? Low yield can result from several factors: insufficient starting material, incomplete lysis (especially for filamentous fungi), DNA loss during column binding (check pH and salt concentration), or elution in insufficient volume. For tough fungi, add a mechanical lysis step before column purification. Also verify that your kit is designed for fungal DNA extraction; some bacterial kits may not efficiently lyse fungal cells.

4. How can I remove polysaccharide contamination from my fungal DNA? Polysaccharide contamination is indicated by a low 260/230 ratio (<2.0) and viscous DNA solution. To remove polysaccharides: (1) Use CTAB extraction buffer, which complexes with polysaccharides and removes them during organic extraction [1]; (2) Perform an additional ethanol precipitation with 0.1 volumes of 3 M sodium acetate; (3) Use a commercial cleanup column designed for polysaccharide-rich samples; (4) Dilute the DNA and test PCR amplification—if successful, proceed with diluted DNA.

References and Further Reading

1. Langsiri N, Meyer W, Irinyi L, et al. Optimizing fungal DNA extraction and purification for Oxford Nanopore untargeted shotgun metagenomic sequencing from simulated hemoculture specimens. 2025. PubMed
2. Yoshioka I, Hayashi C, Endo Y, et al. Detection of fungal contamination on museum books stored under controlled environmental conditions: A discrepancy between culture-based and metagenomic analysis approaches. 2026. PubMed
3. Begum N, Lee S, Almeida M, et al. Enhanced Computational and Experimental Approaches for Comparative Analysis of the Human Mycobiome. 2025. bioRxiv
4. García-Gutiérrez L, Mellado E, Martin-Sanchez PM. Contribution of DNA Metabarcoding to the Environmental Fungal Assessments in Hospitals. 2025. PubMed
5. Rickerts V, Springer J, Gerkrath J, et al. Molecular diagnostics in cancer patients with suspected respiratory mold infections. 2025. PubMed
6. CDC and NIH. Biosafety in Microbiological and Biomedical Laboratories (BMBL), 6th Edition. U.S. Department of Health and Human Services, 2020. CDC
7. National Institutes of Health. NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules. NIH
8. National Center for Biotechnology Information. NCBI Bookshelf: Molecular Biology and Laboratory Methods. NCBI

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