Download Comparison of six extraction techniques for isolation of DNA from

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.
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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-
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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. The ease, speed, and yield of Method 1
make it the preferred method of DNA preparation for
most analytical purposes.
10
Acknowledgements
11
This study was presented at the 97th General Meeting
of the American Society of Microbiology, 4–8 May
1997, Miami Beach, FL, Abstract F-12. This work was
supported in part by grants AI-01411 and CA-18029
from the National Institutes of Health.
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12
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References
1 Beck-Sague CM, Jarvis WR, the National Nosocomial Infections
Surveillance System. Secular trends in the epidemiology of nosocomial fungal infections in the United States, 1980–1990. J Infect
Dis 1993; 167: 1247–51.
2 Morrison VA, Haake RJ, Weisdorf DJ. Non-Candida fungal
infections after bone marrow transplantation: risk factors and
outcome. Am J Med 1994; 96: 497–503.
3 Wald A, Leisenring W, van Burik J, Bowden RA. Epidemiology
of Aspergillus infections in a large cohort of patients undergoing
bone marrow transplantation. J Infect Dis 1997; 175: 1459–66.
4 Birch M, Anderson MJ, Denning DW. Molecular typing of
Aspergillus species. J Hosp Infect 1995; 30: 339–51.
5 Cooper CR, Breslin BJ, Dixon DM, Salkin IF. DNA typing of
isolates associated with the 1988 sporotrichosis epidemic. J Clin
Microbiol 1992; 30: 1631–5.
6 Anderson MJ, Gull K, Denning DW. Molecular typing by random
amplification of polymorphic DNA and M13 southern hy-
 1998 ISHAM, Medical Mycology, 36, 299–303
15
16
17
18
19
303
bridization of related paired isolates of Aspergillus fumigatus. J
Clin Microbiol 1996; 34: 87–93.
Hoffman CS, Winston F. A ten-minute DNA preparation from
yeast efficiently releases autonomous plasmids for transformation
of Escherichia coli. Gene 1987; 57: 267–72.
Raeder U, Broda P. Rapid DNA preparation of DNA from
filamentous fungi. Lett Appl Microbiol 1985; 1: 17–20.
Graham GC, Mayers P, Henry RJ. A simplified method for the
preparation of fungal genomic DNA for PCR and RAPD analysis.
Biotechniques 1994; 16: 48–50.
Glee PM, Russell PJ, Welsch JA, Pratt JC, Cutler JE. Methods
for DNA extraction from Candida albicans. Anal Biochem 1987;
164: 207–13.
Smith GL, Sansone C, Socranksy SS. Comparison of two methods
for the small-scale extraction of DNA from subgingival microoganisms. Oral Microbiol Immunol 1989; 4: 135–40.
Smith GL, Socransky SS, Smith CM. Rapid method for the
purification of DNA from subgingival microorganisms. Oral
Microbiol Immunol 1989; 4: 47–51.
Ausubel F. Preparation of plant DNA using CTAB. Curr Protocols
Molec Biol 1994; Suppl 27: 2.3–2.3.7.
Stewart CN, Via LE. A rapid CTAB DNA isolation technique
useful for RAPD fingerprinting and other PCR applications.
Biotechniques 1993; 14: 748–50.
Kitamura K, Yamamoto Y. Purification and properties of an
enzyme, zymolyase, which lyses viable yeast cells. Arch Biochem
Biophys 1972; 153: 403–6.
Marcilla A, Elorza MV, Mormeneo S, Rico H, Sentandreu R.
Candida albicans mycelial wall structure: supramolecular complexes released by zymolyase, chitinase and beta-mercaptoethanol.
Arch Microbiol 1991; 155: 312–9.
Pringle AT, Forsdyke J, Rose AH. Scanning electron microscope
study of Saccharomyces cerevisiae spheroplast formation. J Bacteriol 1979; 140: 289–93.
Kitamura K, Kaneko T, Yamamoto Y. Lysis of viable yeast cells
by enzymes of Arthrobacter luteus. II. Purification and properties
of an enzyme, zymolyase, which lyses viable yeast cells. J Gen
Appl Microbiol 1974; 20: 323–44.
Scott JH, Schekman R. Lyticase: endoglucanase and protease
activities that act together in yeast cell lysis. J Bacteriol 1980:
142: 414–23.