Letters in Applied Microbiology 1985, 1, 17-20

Rapid preparation of DNA from filamentous fungi

U . R A E D E R & P . B R 0 D A Department of Biochemistry and Applied Molecular Biology,

University of Manchester Institute of Science and Technology, PO B o x 88, Manchester
M 6 0 IQD, U K

Received 1 February 1985 and accepted 1 February 1985

R A E D E R , U . & B R o D A , P . 1985. Rapid preparation of DNA from filamentous

fungi. Letters in Applied Microbiology 1, 17-20.
We describe a general, simple and inexpensive method for the isolation of DNA
from filamentous fungi. Starting from freeze-dried mycelium 0.1415% by weight
can be isolated as high molecular weight DNA suitable for restriction and ligation
in 2 h. The preparation can be done in Eppendorf tubes and allows the processing
of many samples in parallel. We have used the method with the basidiomycetes
Phanerochaete chrysosporium, Coprinus cinereus and the ascomycete Aspergillus
nidulans and others have used it with Trichoderma reesei, Aspergillus niger and for
the isolation of DNA from tomato plants.

There is much interest in the molecular genetics
of filamentous fungi with a major aim being the
development of cloning systems. Difficulties in
the isolation of DNA from filamentous fungi,
caused by fungal nucleases, polysaccharides and
pigments, have led to the development of rather
time-consuming and expensive methods involv-
ing ultracentrifugation (Specht et al. 1982;
Garber & Yoder 1983) or column chromatog-
raphy (Saunders et al. 1984).
As part of our work on the lignin degrading
white rot fungus Phanerochaete chrysosporium
we have developed an extremely easy and rapid
DNA isolation procedure which was shown to
be applicable to other filamentous fungi. It can
be carried out in Eppendorf tubes and is espe-
cially useful for DNA preparations of many dif-
ferent samples in parallel, e.g. in the analysis of
different strains, mutants or transformants.
Materials and methods
C U L T U R E A N D HARVESTING OF F U N G A L
STRAINS
Media for P. chrysosporium (1.5% malt extract
(Oxoid)) and Aspergillus nidulans (g/l : Czapek
Dox liquid medium 33.4; yeast extract 2; casa-
mino acids 6 ) were inoculated with a spore sus-
pension and the cultures were grown at 37°C or
28°C respectively, shaken at 100 rev/min; a
culture of Coprinus cinereus was kindly provided
by R. Haylock. After 1 (P. chrysosporium) or 2 d
(C. cinereus) of growth the material was har-
vested in a sieve and squeezed between filter
papers to remove as much liquid as possible.
(For large scale preparations the material was
transferred from the sieve into a solution of 20
mmol EDTA pH 8 and poured onto a filter
paper in a Buchner funnel connected to a water
pump.) The predried 'cake' is peeled off the filter
paper, folded, immersed into liquid nitrogen
using forceps and lyophilized. The dry material
is ground thoroughly with mortar and pestle,
and transferred with a stiff piece of paper cut to
a round shape into Eppendorf tubes (for up to
50 mg) or into universal bottles with a chloro-
form resistant screw cap (for up to 1 g).
SOLUTIONS FOR THE D N A PREPARATION
Extraction buffer: 200 mmol Tris HCI pH 8.5,
250 mmol NaCI, 25 mmol EDTA, 0.5% SDS;
phenol: e.g. BDH AnalaR, molten at 45"C,
equilibrated with 1 volume of extraction buffer,
stored under buffer at -20°C; RNAse A: e.g.
Sigma RNAse A No 4875, 70 Kunitz Units/mg,
20 mg/ml in 10 mmol Tris HCI ph 7.5, 15 mmol
NaCI, boiled for 10 min, stored at - 20°C.
18 U . Raeder and P . Broda
ISOLATION OF D N A
Freeze dried ground material (50 mg), in an
Eppendorf tube are resuspended in 500 pl
extraction buffer by stirring with a pipette tip.
The slurry is mixed homogeneously with 350 jd
phenol. Then 150 pl chloroform are added and
mixed and the suspension is centrifuged for 1 h
in an Eppendorf centrifuge (13 000 8). The upper
aqueous phase is taken off immediately, trans-
ferred to an Eppendorf tube containing 25 pl
RNAse A solution (becomes turbid) and incu-
bated for 5-10 min at 37°C. The solution is
extracted with 1 volume of chloroform and cen-
trifuged as before but for only 10 min. The
upper phase is transferred into a sterile Eppen-
dorf tube and mixed with about 0.54 volume
isopropanol (250 pl). DNA precipitates visibly
into a lump which is allowed to settle. As much
liquid as possible is taken off with a pipette
without disturbing the precipitate. The tube is
spun for about 5 s and any remaining liquid is
taken off with a drawn out Pasteur pipette. (In
large scale preparations the liquid is simply
decanted.) The pellet is rinsed with 70%
ethanol, dried under vacuum and resuspended
in 100 pl 10 mmol Tris HCI pH 8, mmol EDTA
(sterile).
CSCl- B I S B E N 2 I M I D E GRADIENTS
Bisbenzimide gradients (Miiller and Gautier
1975; Petes et al. 1978; Garber & Yoder 1983;
Raeder & Broda 1984) were performed to
demonstrate that the DNA prepared by the pro-
cedure described here does not contain contami-
nants that interfere with the fractionation that
depends upon the GC content. These gradients
are not part of the actual isolation procedure.
They were prepared with DNA extracted in the
way described above from 0.5 g of dry material,
in 8.3 ml 50 mmol Tris HCl pH 8, 5 mmol
EDTA, 9.6 g CsCl, plus 1 ml of a 1 mg/ml bis-
benzimide solution (Sigma, Hoechst dye N o
33258, dissolved in H,O) by centrifugation in a
Beckman Ti50 rotor at 34 000 rev/min (75 000 g)
for 65 h at 17°C. Alternatively these gradients
P A

a b
Fig. 1. DNA of Phonerochaeta chrysosporium (P), Coprinus cinereus (C) and Aspergillus nidulans (A) after electro-
phoresis in a 0.3% agarose gel run for 14 h at 3 V/cm at 4°C. Lanes 1 and 2 contain marker DNAs (Lane 1:
lgtWES T5-622 cut with EcoRI. 21.3, 14.3 kb; Lane 2: lc1857, 50 kb for the monomer). (b) DNA prepared
according to the procedure described here of Sporotrichum puluerulentum, P . Chrysosporium, C . cinereus and A .
nidulans uncut (3, 4, 5, 6), DNA of P. chrysosporium, C . cinereus and A . nidulans after restriction with BamHI (7, 8,
9; 3.5 pg DNA after incubation for 1 h at 37°C with 1.2 u BamHi per pg of DNA) and after religation (10, 11, 12;
7 pg DNA after incubation for 16 h at 7°C with 0.9 Weiss Units T4 Ligase). Lanes 1 and 2 show the very small
amounts of DNA which did not precipitate with isopropanol in preparations from 20 mg dry material of S.
puluerulentum (1) and P . chrysosporium (2), but did then precipitate from the supernatant liquid with 2 volumes of
ethanol. The heavy bands migrating more slowly than the very faint DNA bands do not show the typical orange
fluorescence of ethidium bromide-stained DNA but a dark red fluorescence, and they were only observed with S .
puluerulentum and P. chrysosporium. Lane 13 contains lcI857 DNA cut with Hind111 as size marker. (c) Fraction-
ation in CsC1-bisbenzimide gradients of DNA, prepared from 0.5 g dry weight as described here, into mitochon-
drial DNA (upper band), chromosomal DNA (lower band), and in the case of P . chrysosporium, a third satellite
band (above the main chromosomal band) containing ribosomal DNA (Raeder & Broda 1984).
 D N A from jilamentous fungi
may be prepared in a Beckman VTi65 vertical
rotor with 4.15 ml DNA solution, 4.41 g CsCl
and 0.45 ml bisbenzimide solution by centrifu-
gation overnight at 49 000 rev/min.
Results
Resuspension of the DNA gives clear, colour-
less, often viscous solutions with spectrophoto-
metric ratios of 0.D.260:0.D.28,between 1.9
and 2.0. The DNA yields are around 0.12% of
mycelial dry weight for P . chrysosporium and C.
cinereus and 0.15% for A . nidulans. The DNA is
of high molecular weight; Fig. l a shows that it
migrates with similar mobility to undigested i
DNA in agarose gel electrophoresis.
The DNA is cut efficiently with restriction
endonucleases (tested with EcoRI, BamHI,
HindIII, PstI, SalI and Sau3A) and is ligatable.
Figure Ib shows DNA after incubation with
1.2 p BamHI per 1 pg of DNA for 1 h at 37°C:
DNA from both C. cinereus and A . nidulans was
restricted to completion (as judged from the
well-defined hybridization signals obtained from
a Southern blot of this gel after hybridization
with a labelled ribosomal DNA probe of P.
chrysosporium). Restriction of the P . chryso-
sporium DNA was only partial in this experi-
ment (Fig. Ib), but complete digests were
obtained after 90 min incubation. Comparison
of the size distribution of restricted DNA (lanes
7, 8, 9) and religated DNA (lanes 10, 11, 12)
shows that this DNA can be ligated efficiently.
We have also used P . chrysosporium DNA pre-
pared by this method for construction of 1 gene
banks with similar efficiency to those obtained
with CsC1-gradient-purified DNA.
Figure l c shows that DNA prepared accord-
ing to our procedure fractionates in CsCI-
bisbenzimide gradients to give mitochondrial
DNA (upper bands), chromosomal DNA (lower
bands) and in the case of P . chrysosporium a
third band (above the lowest chromosomal
band) containing ribosomal DNA (Raeder &
Broda 1984).
Discussion
The main difficulty in obtaining DNA from P .
chrysosporium suitable for restriction and liga-
tion was caused by polysaccharides which could
only be removed by such relatively inconvenient
methods as DE52 column chromatography,
sedimentation by ultracentrifugation or in CsCl-
19
ethidium bromide gradients. The problem was
overcome by a combination of two steps:
(1) a 1 h long centrifugation at 13000 g (or
more) after the phenol chloroform extraction to
sediment high molecular weight polysaccharide
onto the interphase of debris and denatured
protein. (In large scale preparations a fixed
angle rather than a swing out rotor should be
used for shorter sedimentation distances.)
(2) selective 'and quantitative precipitation of
DNA with 0.54 volumes isopropanol (Marmur
1961; Cryer et al. 1975) in the form of one big
aggregate, and immediate removal by pipette of
the liquid which contains residual pigment and
residual slowly precipitating polysaccharide.
The quantitative aggregation of DNA upon
isopropanol precipitation depends on a high
DNA concentration (around 100 pg/ml due to
the very small extraction volume of 100 p1/10
mg mycelial dry weight) and on its high molecu-
lar weight. Therefore Vortex mixing or the use
of old phenol should be avoided as they reduce
the molecular weight and lead to more dis-
persed DNA precipitation. Precipitation with 1
volume of isopropanol or 2 volumes of ethanol
result in co-precipitation of contaminants and
reduced restrictability of the DNA.
The yield of high molecular weight DNA of
0.1-0.15% of the mycelial dry weight is con-
siderably greater than in other methods (Specht
et al. 1982; Garber & Yoder 1983), probably due
to the very few preparation steps. Long extrac-
tion times of 1-3 d as recommended by Specht
et al. (1982) were not found to be necessary. The
DNA yield did not depend significantly on the
duration of the extraction but on the small par-
ticle size of the ground material. Fungal nucle-
ases were reliably eliminated by extraction with
phenol.
This work was part of a programme supported
jointly by British Petroleum's Venture Research
Unit and the Agriculture and Food Research
Council.
References
CRYER, D.R., ECCLESHALL, R. & MARMUR, J. 1975 Iso-
lation of yeast DNA. Methods in Cell Biology 12,
39-44.
GARBER, R.C. & YODER,O.C. 1983 Isolation of DNA
from filamentous fungi and separation into nuclear,
mitochondrial, ribosomal and plasmid components.
Analytical Biochemistry 135, 416422.
20 U . Raeder and P . Broda
MARMUR,J. 1961 A procedure for the isolation of
deoxyribonucleic acid from microorganisms.
Journal of Molecular Biology 3, 208-218.
MULLER,W. & GAUTIER, F. 1975 Interaction of het-
eroaromatic compounds with nucleic acids. A T-
specific non-intercalating DNA ligand. European
Journal of Biochemistry 54, 385-394.
PETS, T.D., HEREFORD, L.M. & BOTSTEIN, D. 1978
Simple Mendelian inheritance of the reiterated
ribosomal DNA of yeast. Cold Spring Harbor Sym-
posia on Quantitative Biology, 42, Part 11, 1201-
1207.
RAEDER,U. & BRODA,P. 1984 Comparison of the
lignin degrading white rot fungi Phanerochaete
chrysosporium and Sporotrichum pulverulentum at
the DNA level. Current Genetics 8,499-506.
SAUNDERS, G., ROGERS, M.E., ADLARD, M.W. & HOLT,
G. 1984 Chromatographic resolution of nucleic
acids extracted from Penicilliurn chrysogenum.
Molecular and General Genetics 194,343-345.
SPECHT,C.A., D~Russo, C.C., NOVOTNY,P.C. &
ULLRICH,R.C. 1982 A method for extracting high-
molecular-weight deoxyribonucleic acid from fungi.
Analytical Biochemistry 119, 158-163.