A simple method for extraction of fungal genomic DNA

Letters in Applied Microbiology 2000, 30, 53–56

A simple method for extraction of fungal genomic DNA T.H. Al-Samarrai and J. Schmid Institute of Molecular BioSciences, Massey University, Palmerston North, New Zealand 2261/99: received 8 July 1999 and accepted 25 September 1999 T . H . A L -S AM A RR AI A ND J. S CH MI D . 2000. We have developed a new, simple and effective method for extraction of fungal genomic DNA. The initial steps involved suspension of freeze-dried mycelium in buffer containing sodium dodecyl sulphate, detachment of DNA from polysaccharides by mild shearing, NaCl precipitation of polysaccharides and protein, chloroform extraction and ethanol precipitation. The ethanol precipitate was then subjected to a second round of mild shearing, NaCl precipitation, chloroform extraction and ethanol precipitation. The procedure required approximately 1 h to perform. The method yielded 8–32 mg of high molecular weight DNA per 30 mg of freeze-dried mycelium when tested on six fungal species: Aspergillus niger, A. flavus, Fusarium graminarum, Neotyphodium lolii, Penicillium citrinum and Rhizopus nigricanes. The DNA was digestible with EcoRI, HindIII, SalI and BamHI. For the slow-growing N. lolii, a modification of the method was developed that removed the agar residue from colonies grown on agar plates by centrifugation at 13 000 rev min−1 in the presence of CsCl. The modified method yielded 1·5–2 mg of high molecular weight DNA per colony.

INTRODUCTION

It is widely accepted that the extraction of pure, high molecular weight DNA from fungi is difficult. We found that DNA extraction from Neotyphodium lolii (Christensen et al. 1993) by the methods of Raeder and Broda (1985) and Byrd et al. (1990) yielded DNA that was not digested by restriction enzymes nor amplified by Taq polymerase. This was attributed mainly to contamination by polysaccharides produced either by the fungus or transferred to liquid cultures with the innoculum from agar plates. Contamination by polysaccharides has also been encountered and circumvented in the preparation of DNA from Gram-negative bacteria. We therefore modified the procedure developed by Chen and Kuo (1993) for use in the preparation of DNA from filamentous fungi. MATERIALS AND METHODS Fungal strains

Neotyphodium lolii strain Lp19 was obtained from AgResearch Grasslands, Palmerston North, New Zealand. AsperCorrespondence to: Taha H. Al-Samarrai, Institute of Molecular BioSciences, Massey University, Private Bag 11222, Palmerston North, New Zealand (e-mail: [email protected]). © 2000 The Society for Applied Microbiology

gillus niger (no. 271), A. flavus (no. 8), Fusarium graminarum (no. 177), Penicillium citrinum (no. 264) and Rhizopus nigricanes (no. 100) were obtained from the fungal collection of the Institute of Molecular BioSciences, Massey University, New Zealand. Preparation of fungal mycelia

Freeze-dried mycelium from submerged cultures of N. lolii was prepared as follows. Colonies of the fungus (diameter 25–30 mm), grown on potato dextrose agar at 22 °C for 3–4 weeks, were removed from the surface of the agar with a sterile scalpel and transferred to a sterile Petri dish. Agar was removed from the colony with a sterile spatula and the mycelium was cut into small pieces (less than 0·5 mm) with a sterile scalpel. Complete medium (Pontecorvo et al. 1953), 100 ml in a 150-ml flask, was inoculated with these pieces of mycelium and incubated for 10 d at 22 °C with shaking at 200 rev min−1. The mycelium was harvested by filtration through Whatman filter paper, washed with distilled water, freeze-dried and stored at −20 °C. Freeze-dried N. lolii mycelium was also prepared from colonies grown on potato dextrose agar as described above. A single colony was removed from the agar with a scalpel, freed from traces of agar, suspended in 1 ml of distilled water

54 T .H . A L -S AM A RR AI A ND J. S CH MI D

in an Eppendorf tube and fragmented by pipetting. CsCl, 1 g, was added and following centrifugation at 13 000 rev min−1 for 10 min, the bulk of the mycelium was removed from the surface of the CsCl solution, leaving behind the agar at the bottom and a few mycelial stands dispersed in the solution. After washing twice with distilled water in an Eppendorf tube, the mycelium was freeze-dried and stored at −20 °C. One colony yielded approximately 15 mg of dry mycelium. Freeze-dried mycelium of all the other fungi was prepared from cultures grown in liquid complete medium as described above for N. lolii, except that the medium was inoculated with a spore suspension obtained by washing colonies, grown on potato dextrose agar, with 5 ml of sterile saline. The mycelium was harvested after 5 d.

DNA was washed three times with 70% ice-cold ethanol, dried and dissolved in 50 ml TE buffer (10 mmol/l Tris-HCl, 0·1 mmol/l EDTA pH 7·8) and stored at −20 °C. Other DNA extraction methods

DNA was also prepared from freeze-dried mycelium of N. lolii by the method of Raeder and Broda (1985) and which

Extraction of fungal DNA

Thirty mg of freeze-dried mycelium was ground to a fine powder in an Eppendorf tube in liquid nitrogen using a pre-cooled pestle (Eppendorf no. 0030120973). The ground mycelium was resuspended and lysed in 500 ml of lysis buffer (40 mmol/l Tris-acetate, 20 mmol/l sodium acetate, 1 mmol/l EDTA, 1% w/v SDS pH 7·8) (Lerner and Model 1981) by pipetting with a Gilson P 1000 pipetman (set to 1000 ml) until the viscosity of the suspension was significantly reduced and the formation of froth indicated the detachment of DNA from polysaccharides. For most fungi five cycles of gentle pipetting were sufficient; however, for N. lolii at least 30 cycles of vigorous pipetting was necessary. RNAse A (2 ml of 10 mg/ml; Sigma, St Louis, MO, USA) was added and the mixture was incubated for 5 min at 37 °C. To facilitate the precipitation of most polysaccharides, protein and cell debris, 165 ml of 5 mol/l NaCl solution was added and the components mixed by inverting the tube several times. The suspension was centrifuged at 13 000 rev min−1 for 20 min at 4 °C, the supernatant was immediately transferred to a fresh tube and 400 ml of chloroform and 400 ml of phenol were added. The solution was mixed by gently inverting the tube until the solution became milky (usually 50 times). After centrifugation for 20 min, the aqueous phase was removed and extracted with an equal volume of chloroform. The DNA in the aqueous supernatant was precipitated with two volumes of 95% ethanol. To free the DNA from polysaccharide the precipitate was resuspended in 500 ml of lysis buffer and mixed by gentle pipetting. Then 165 ml of 5 mol/l NaCl was added and the tube gently inverted several times. The suspension was then chloroform-extracted as described above. Usually, after centrifugation for 10 min, the aqueous phase was clear and the DNA was precipitated with 95% ethanol. On rare occasions the aqueous phase was still cloudy, in which case it was re-extracted with one volume of chloroform before the DNA was precipitated. The precipitated

Fig. 1 Molecular weight and digestibility of DNA prepared, from N. lolii grown in submerged culture, by different methods. Lanes 1 (digested with SalI) and 2 (uncut) DNA prepared by the method of Byrd et al. (1990). Lanes 3 (digested with SalI) and 4 (uncut) DNA prepared by the method described in this paper. Lanes 5 (digested with SalI) and 6 (uncut) DNA prepared by the method of Raeder and Broda (1985). Lane 7 1kb ladder

Table 1 Yield of DNA from freeze-dried mycelium for different fungi grown in submerged culture — ––––––––––––––––––––––––––––––––––––––––––––––––––––– Mean yield (mg per Fungal species mg mycelium) 2 SD — ––––––––––––––––––––––––––––––––––––––––––––––––––––– N. lolii (Lp19) 0·83 2 0·20 A. flavus 0·34 2 0·63 A. niger 0·39 2 0·08 R. nigricanes 0·45 2 0·11 P. citrinum 0·87 2 0·19 F. graminarum 0·38 2 0·11 — ––––––––––––––––––––––––––––––––––––––––––––––––––––– The data are the average of five determinations.

© 2000 The Society for Applied Microbiology, Letters in Applied Microbiology 30, 53–56

E XT RA C TI ON O F F UN G AL DN A 55

Fig. 2 Molecular weight and digestibility of fungal DNA prepared with the new method from mycelium grown in submerged culture. Lanes 2, 7, 12, 17, 22 and 27 show DNA from N. lolii, A. niger, A. flavus, P. citrinum, F. graminarum and R. nigricanes, respectively, incubated under the same condition as used for restriction digests, but in the absence of restriction enzymes. Lanes 3–6 show DNA from N. lolii, lanes 8–11 DNA from A. niger, lanes 13–16 DNA from A. flavus, lanes 18–21 DNA from P. citrinum, lanes 23–26 DNA from F. graminarum and lanes 28–31 DNA from R. nigricanes, cut with EcoRI, HindIII, SalI and BamHI, respectively. Lanes 1 and 32 show XV (Boehringer Mannheim) molecular weight marker

involved phenol/chloroform extraction of powdered mycelium in buffer (200 mmol/l Tris-HCl, 250 mmol/l NaCl, 25 mmol/l EDTA, 0·5% w/v SDS pH 8·5) and the method of Byrd et al., (1990) which involved phenol/chloroform extraction of the supernatant following proteinase K treatment of powdered mycelium in buffer (50 mmol/l Tris-HCl, 150 mmol/l EDTA, 1% w/v sodium lauryl sarcosine pH 8·0). Determination of DNA yield

DNA was determined by a fluorometric assay, using Hoechst dye 33258 (Brunk et al. 1979) and a TKO 100 Mini Fluorometer (Hoeffer). Digestion with restriction enzymes

Each digestion (1 h at 37 °C) contained, in a total volume of 20 ml, 1–2 mg DNA, 1 mg bovine serum albumin, 20 U of restriction enzyme and 2 ml of the 10× buffer supplied with the enzyme (Boehringer Mannheim, Mannheim, Germany). To assess autodegradation of DNA, a DNA sample was also incubated under the same conditions but in the absence of restriction enzyme. The entire digest was loaded onto a 0·5% w/v agarose gel containing TBE buffer (89 mmol/l Tris, 89 mmol/l boric acid, 2·5 mmol/l EDTA pH 8·3) and electrophoresis performed at 1·0 V cm−1 for 20 h at 22 °C.

freeze-dried mycelium and by the method of Raeder and Broda (1985) resulted in DNA that could not be cut by SalI (Fig. 1). An extraction method was therefore developed to obtain good yields of pure, high molecular weight DNA. For several fungi, grown in submerged culture, the extraction method yielded 0·34–0·87 mg DNA mg−1 freeze-dried mycelium. Average yields are listed in Table 1. The DNA dissolved easily in buffer and was of high molecular weight (Fig. 2). DNA from all fungi was cut efficiently with EcoRI, HindIII, SalI and BamHI (Fig. 2). High molecular weight DNA, which was digestible with restriction enzymes, was also obtained from N. lolii mycelium taken directly from agar plates although the yield was only 0·13 mg mg−1 freeze-dried mycelium. In summary the present method gives good yields of high molecular weight, digestible DNA across a wide range of fungi.

ACKNOWLEDGEMENTS

We thank M. Christensen and A. Lindsey for supplying fungal cultures. We thank P. Sullivan and P. Farley for their invaluable advice in the preparation of this manuscript. This work was supported by grant MAU 403 from the New Zealand Foundation for Research, Science and Technology.

REFERENCES RESULTS AND D ISCUSSION

Attempts to obtain DNA from freeze-dried N. lolii mycelium, prepared from submerged cultures, by the method of Byrd et al. (1990) resulted in yields of only 0·002 mg DNA mg−1

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