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Medical Mycology 1998, 36, 299–303 Accepted 21 May 1998 Comparison of six extraction techniques for isolation of DNA from filamentous fungi J.-A. H. VAN BURIK,∗† R. W. SCHRECKHISE,∗ T. C. WHITE,‡§ R. A. BOWDEN∗† & D. MYERSON¶∗∗ Programs in ∗Infectious diseases and ¶Pathology, Fred Hutchinson Cancer Research Center; ‡Seattle Biomedical Research Center; the Divisions of †Allergy and Infectious Diseases, §Pathobiology, and ∗∗Pathology, University of Washington, Seattle, Washington, USA Filamentous fungi have a sturdy cell wall which is resistant to the usual DNA extraction procedures. We determined the DNA extraction procedure with the greatest yield of high quality fungal DNA and the least predilection for crosscontamination of equipment between specimens. Each of six extraction methods was performed using Aspergillus fumigatus hyphae. The six methods were: (1) glass bead pulverization with vortexing; (2) grinding with mortar and pestle followed by glass bead pulverization; (3) glass bead pulverization using 1% hydroxyacetyl trimethyl ammonium bromide (CTAB) buffer in a water bath sonicator; (4) water bath sonication in CTAB buffer; (5) grinding followed by incubation with CTAB; and (6) lyticase enzymatic cell lysis. Genomic DNA yields were measured by spectrophotometry and by visual reading of 2% agarose gels, with shearing assessed by the migration of the DNA on the gel. Genomic fungal DNA yields were highest for Method 1, followed by Methods 5≅2>3≅4≅6. Methods 2 and 5, both of which involved grinding with mortar and pestle, led to shearing of the genomic DNA in one of two trials each. We conclude that the use of glass beads with extended vortexing is optimal for extraction of microgramme amounts of DNA from filamentous fungal cultures. Keywords DNA extraction, DNA isolation, filamentous fungi, fungal cell breakage, glass beads, lyticase Introduction With the current growth in the numbers of immunocompromised hosts, the prevalence of invasive fungal infections has increased [1–3]. Concurrent with a rise in the rate of fungal infections, the potential for applications of molecular biology to these infections has also increased, such as strain typing to determine the relatedness of fungal isolates from outbreaks among infected patients [4–6]. Filamentous fungi have a sturdy cell wall which is resistant to standard DNA extraction Correspondence: Jo-Anne van Burik, 1100 Fairview Avenue North D3-100, P.O. Box 19024, Seattle WA 98109-1024, USA. Tel. (206) 667 5972; Fax. (206) 667 4411; E-mail: [email protected]. 1998 ISHAM procedures for yeast and bacteria. To reduce the cost, labour and time involved in molecular typing experiments, isolation of the fungal DNA would ideally be performed by processing one plate of fungal growth rather than using conidia and hyphal fragments from plate cultures to seed larger volume broth cultures. Thus, isolation of DNA for molecular typing methods has required large numbers of cells to achieve the relatively large starting amounts of DNA. In the present study, DNA yields from various extraction methodologies have been compared systematically to determine the DNA extraction procedure with the greatest yield of fungal DNA having little protein content, and the least predilection for crosscontamination by reusable equipment. 300 van Burik et al. Materials and methods Fungal cultures A pure culture of Aspergillus fumigatus, obtained from a patient with invasive pulmonary aspergillosis, was grown in a 100 ml volume of RPMI 1640 (American Biorganics; Niagara Falls, NY, USA) for 7 days at 37 °C. The mycelial mat was separated from the culture supernatant by vacuum filtration through a sterile 0·2 lm pore size disposable filter (Nalgene, Rochester, NY, USA), followed by continuous vacuum until dry. The mycelia were stored frozen at −70 °C until ready for use. The mycelial mat was cut into sections containing approximately 30–100 mg for use in testing each extraction method. After the six extraction methods were tested, various other fungi stocked by the laboratory were also tested for compatibility with the ‘optimal’ method. Visual estimates of DNA made from the agarose gels correlated with the amounts of raw DNA calculated using readings from the spectrophotometer. Extraction method 1: glass beads and vortexing Using minor modifications of a previously published method as described below [7], mycelium was transferred to a locking microcentrifuge tube using a sterile spatula and suspended in 400 ll extraction buffer [2% Triton X-100, 1% SDS, 100 m NaCl, 10 m Tris-Cl (pH 8·0), 1 m EDTA]. Lysis of the mycelium was achieved by the addition of 500 mg acid-washed 0·4–0·6mm diameter glass beads (Sigma, St Louis, MO, USA), 400 ll phenol/chloroform/iso-amyl alcohol (Phe/Chl/ IAA) (24:24:1), and continuous vortexing for 30 min at the highest intensity setting [8] utilizing a vortexGenie 2 (Fisher Scientific, Santa Clara, CA, USA) fitted with a 6-inch platform head and microtube insert capable of holding up to 60 microcentrifuge tubes. Vortexing with Phe/Chl/IAA was performed in a fume hood under continuous supervision so that the vortexer remained centred and the tubes locked. The aqueous layer was removed and re-extracted with an equal volume of Phe/Chl/IAA (24:24:1) twice, an equal volume of Chl/IAA (24:1) once, and precipitated with 0·1 volume 10 ammonium acetate followed by 2·0 volume 100% ethanol. The resulting fungal DNA pellet was resuspended in 100 ll 10 m Tris (pH 8·0), 1 m EDTA, and 1 ll 500 lg/ml Rnase (Boehringer Mannheim, Indianapolis, IN, USA). The digestion was incubated at 37 °C for 1 h. To remove residual cellular debris, the tube was spun at high speed in a microcentrifuge for 10 min (13 000 g), and the supernatant transferred to a new tube. Extraction method 2: grinding, glass beads and bead beating Following a previously published method [8], mycelium was transferred to an autoclaved, pre-cooled mortar, frozen with liquid nitrogen, ground to a fine powder and placed in a microcentrifuge tube. Further pulverizing was accomplished by combining the powdered mycelia with 500 mg of glass beads in a microfuge tube and vortexing at full speed for 30 min. The ground material was suspended in 500 ll buffer [200 m Tris (pH 8·5), 250 m NaCl, 25 m EDTA and 0·5% SDS] and placed on a rocker platform for 30 min. Nucleic acids were extracted twice with 500 ll Phe/Chl/IAA (24: 24:1), once with 500 ll Chl/IAA (24:1) and precipitated with 0·7 volume isopropanol. RNA and cellular debris were removed as described above in Method 1. Extraction method 3: glass beads, CTAB and sonication Mycelium was suspended in 600 ll extraction buffer [1% CTAB (hydroxyacetyl trimethyl ammonium bromide) (Acros, Pittsburgh, PA, USA), 1·4 NaCl, 100 m Tris (pH 8·0), 20 m EDTA] in a microcentrifuge tube [9]. Lysis of the mycelia was achieved by sonication for 40 min at 55 °C and 47 kHz in a Bransen 2200 water bath sonicator (Bransen Ultrasonics Corp., Danbury, CT, USA) in the presence of 500 mg of glass beads. The supernatant was transferred to a new tube after centrifugation for 5 min at high speed in a microcentrifuge. DNA was further extracted using Phe/Chl/ IAA and RNA and cellular debris removed as per Method 1. Extraction method 4: CTAB and sonication Method 4 was identical to Method 3, except that glass beads were not added during sonication. Extraction method 5: grinding and CTAB Mycelium was transferred to a pre-cooled, sterile mortar and pestle, frozen with liquid nitrogen and ground to a fine powder, then transferred to a microcentrifuge tube [9]; 600 ll of 1% CTAB extraction buffer was used to wash the mortar and pestle and suspend the ground mycelia. The tube was incubated on ice for 1 h. DNA was further extracted using Phe/Chl/IAA and RNA and cellular debris removed as per Method 1. Extraction method 6: lyticase Mycelium was transferred to a microcentrifuge tube, suspended with >500 ll lysis buffer [50 m Tris 1998 ISHAM, Medical Mycology, 36, 299–303 DNA extraction techniques for filamentous fungi 301 Table 1 The amount of raw DNA recovered from each extraction method as measured by spectrophotometry Start weight End weight 260/280 Ratio % Yield∗ % Maximum yield Glass beads/vortex 110 mg 30 mg 81·8 lg 21·6 lg 1·725 1·628 0·074 0·072 100 97 2 Grinding/glass beads/bead beater 100 mg 40 mg 45·3 lg 10·6 lg 1·554 0·770 0·045† 0·026 61 35 3 Glass beads/CTAB/sonication 60 mg 30 mg 6·2 lg 4·2 lg 1·392 1·462 0·010 0·014 14 19 4 CTAB/sonication 100 mg 30 mg 11·5 lg 3·2 lg 1·478 1·446 0·012 0·011 16 14 5 Grinding/CTAB 120 mg 40 mg 15·0 lg 15·5 lg 1·722 1·677 0·013 0·039† 18 52 6 Lyticase 100 mg 10 mg 10·4 lg 8·3 lg 1·643 1·564 0·010 0·021 14 28 Number Method 1 ∗Yield=weight of extracted DNA/weight of wet mycelia at start. †Shearing of the DNA visible on the agarose gel. (pH 7·6), 1 m EDTA, 20% 2-mercaptoethanol], to which >300 units lyticase (Sigma) were added. The digestion was allowed to incubate for 3 h at 37 °C, then the nuclei were lysed by the addition of 100 ll 10% SDS while incubated at 65 °C for 20 min. Protein and cellular debris were precipitated by the addition of 200 ll 5 potassium acetate and incubated on ice for 25 min. The tube was centrifuged for 10 min at high speed, the supernatant transferred to a new tube, and 600 ll isopropanol was added. The tube was incubated on ice for 30 min and cold-centrifuged for 30 min at high speed. The pellet was washed with 70% ethanol, then resuspended in buffer (10 m Tris, 1 m EDTA, and 500 lg ml−1 RNase, as described in Method 1. Detection of extracted DNA DNA was quantitated in a spectrophotometer (Spectronic 1001, Milton Roy Company, Rochester, NY, USA) using 1:30 dilutions. The raw yield was calculated as the weight of extracted DNA divided by the weight of the mycelia at start. Purity was estimated with the 260/280 ratio. Shearing was assessed by a visual reading of 5 ll run on 2% agarose gels. Results The starting mycelial weight and the amount of DNA recovered from each extraction method are listed in Table 1. Fungal DNA yields were highest for glass bead pulverization with vortexing, but all methods 1998 ISHAM, Medical Mycology, 36, 299–303 provided enough DNA for multiple Southern hybridizations or PCR assays. Grinding with mortar and pestle led to yields half that of glass beads. The latter method led to shearing of the genomic DNA, although the shearing assessment was limited by the fact that the range of separation for 2% agarose gels is 0·1–2 kbp of linear DNA molecules. The last four methods yielded five- to 10-fold smaller amounts of DNA when compared to Method 1. Figure 1 is an ethidium bromide-stained 2% agarose gel of 1/20th of the raw DNA yield from each of the six extraction methods. All lanes are free of contaminating RNA. Although the yield was relatively high, Method 2 led to sheared DNA (the range of separation from a 2% agarose gel is limited and may not show differences needed for genomic Southerns or library construction). Method 5 also led to shearing of the genomic DNA in one of two trials (only one trial is shown in Fig. 1). Therefore, genomic fungal DNA yields were highest for Method 1, followed by Methods 5>6>4≅3, but Method 5 occasionally led to shearing of DNA. Combining the results of both trials, fungal DNA yields were highest for Method 1, followed by Methods 5≅2>3≅4≅6. In addition to A. fumigatus, DNA has been successfully isolated using Method 1 from other fungi of the following species: Absidia, Acremonium, Alternaria, Aspergillus flavus, A. nidulans, A. niger, A. terreus, Aureobasidium pullulans, Bipolaris, Blastomyces dermatitidis, Candida albicans, C. glabrata, C. krusei, C. parapsilosis, Chaetomium, Chrysosporium, Clado- 302 van Burik et al. Fig. 1 Ethidium bromide-stained 2% agarose gel of 1/20th of the resulting A. fumigatus DNA from each of the six extraction methods. Lane 1, uX174/HaeIII DNA ladder has 10 bands visible, corresponding with the following numbers of base pairs: 1353, 1078, 872, 603, 310, 281/271, 234, 194 and 118. The uppermost band of uX174/HaeIII represents 100 ng DNA. The remaining lanes are labelled with the extraction method number. Sample 2 was sheared to a very small molecular weight. The lanes are numbered according to extraction method: (1) glass beads, vortexing; (2) grinding with mortar/pestle, glass beads, bead beater; (3) glass beads, CTAB buffer, sonication; (4) CTAB buffer, sonication; (5) grinding with mortar/pestle, CTAB buffer incubation; (6) enzymatic lysis using lyticase. phialophora carrionii, Cryptococcus neoformans, Cunninghamella, Curvularia, Drechslera, Fonsecaea pedrosoi, Fusarium solani, Hortaea werneckii, Lecythophora, Malassezia furfur, Microsporum canis, M. gypseum, Mucor indicus, Paecilomyces, Penicillium, Phoma, Pichia mrakii, Prototheca wickerhamii, Pseudallescheria boydii, Rhizomucor pusillus, Rhizopus arrhizus, Rhodotorula rubra, Scopulariopsis, Sepedonium, Sordaria macropoia, Sporothrix schenckii, Trichophyton rubrum, Ulocladium, Verticillium and Zygorhynchus (data not shown). DNA was also isolated using Method 1 from mycelia that grew on solid medium but did not develop the fruiting structures necessary for identification (sterile mycelia). The amount of DNA isolated was approximately equivalent between species. Discussion Traditional methods of fungal DNA extraction have included physical disruption, homogenization, sonication, French press or glass beads [10]. DNA isolation protocols for the breakage of yeast cells involve vortexing with glass beads in a detergent solution and separating nucleic acids from protein by phenol/chloroform extraction [7]. When preparing S. cerevisiae DNA, 3 min of vortexing is generally sufficient to break the cells yet reduce shearing of DNA. The average yield is >20 lg DNA from a 10-ml stationary-phase culture. The application of glass beads and vortexing to cultures of filamentous fungi has not been described. Historically, the non-ionic detergent hydroxyacetyl trimethyl ammonium bromide (CTAB) has been used to extract DNA from bacteria [11,12] and plants [13, 14]. In a study of 1% CTAB, this detergent yielded enough genomic fungal DNA for random-amplified polymorphic DNA analysis of fungal specimens [9]. Transfer of the minutest amount of fungal DNA by reusable equipment or reagents can lead to detrimental and confounding cross-contamination of specimens in applications such as molecular typing. Therefore, enzymatic methods of fungal cell wall disruption could become a preferred extraction method for obtaining consistent release of fungal DNA because all equipment, supplies and reagents are disposable. In the 1970s, snail gut enzyme was the prototype enzyme used for fungal cell wall lysis, but the preparation was variable in activity from batch to batch [15]. Currently available enzymes include b-1,3-glucanases or chitinases [16]. Beta-1,3-glucanase enzymes hydrolyze glucose polymers at b-1,3-glucan linkages to release laminaripentaose from fungal cell walls. The result is a fungal spheroplast which is osmotically unstable [17]. Once the spheroplast is formed, a nuclear lysis agent such as sodium dodecyl sulfate is used to release fungal DNA. b-1,3-glucanase products include zymolyase (ICN Pharmaceuticals, Costa Mesa, CA, USA), a natural b-1,3-glucanase purified from a submerged culture of Arthrobacter luteus in the fermentation of yeast [18], and lyticase (Sigma, St Louis, MO, USA), a synthetic equivalent [19]. Use of lyticase avoids the impurities found in zymolyase, such as b-1,3-gluconase, protease, mannanase, amylase, xylanase, phosphatase and trace DNAse. By testing the yields of six methodologies, we have shown that glass bead pulverization with extended vortexing, Method 1, produces the highest and most reproducible yield of filamentous fungal DNA. This DNA extraction procedure took 2–3 h from the beginning of processing until the DNA was ready for use, in contast to enzymatic digestion and isopropanol precipitation which can take 5–8 h. When several fungal isolates were extracted in parallel, little increase in the processing time was noted for Method 1. The major advantage of glass bead pulverization with extended vortexing is that reusable equipment is not required 1998 ISHAM, Medical Mycology, 36, 299–303 DNA extraction techniques for filamentous fungi and hence there is a low risk for transfer of DNA between specimens. The DNA is suitable for PCR amplification (data not shown). This method of extraction can be useful for strain typing experiments, as the amount of fungus that can be obtained from scraping a culture slant is the starting amount used for these experiments. 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