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A simple method of genomic DNA extraction suitable for analysis of bulk

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Article  in  Letters in Applied Microbiology · July 2010

DOI: 10.1111/j.1472-765X.2010.02867.x · Source: PubMed

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 Letters in Applied Microbiology ISSN 0266-8254

ORIGINAL ARTICLE

A simple method of genomic DNA extraction suitable for

analysis of bulk fungal strains
Y.J. Zhang1,2, S. Zhang1, X.Z. Liu2, H.A. Wen2 and M. Wang3
1 School of Life Sciences, Shanxi University, Taiyuan, China
2 Key Laboratory of Systematic Mycology and Lichenology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China
3 School of Plant Sciences & Technology, Agriculture and Animal Husbandry College of Tibet, Nyingchi, China

Keywords
DNA extraction, fungi, internal transcribed
spacer (ITS) region, Ophiocordyceps sinensis,
PCR amplification, thermolysis.
Correspondence
Xing Zhong Liu, Institute of Microbiology,
Chinese Academy of Sciences, No.3, 1st West
Beichen Road, Chaoyang District, Beijing
100101, China.
E-mail: liuxz@im.ac.cn (X.Z. Liu)
2010 ⁄ 0284: received 17 February 2010,
revised and accepted 30 April 2010
doi:10.1111/j.1472-765X.2010.02867.x
Abstract
Aims: A simple and rapid method (designated thermolysis) for extracting geno-
mic DNA from bulk fungal strains was described.
Methods and Results: In the thermolysis method, a few mycelia or yeast cells
were first rinsed with pure water to remove potential PCR inhibitors and then
incubated in a lysis buffer at 85C to break down cell walls and membranes.
This method was used to extract genomic DNA from large numbers of fungal
strains (more than 92 species, 35 genera of three phyla) isolated from different
sections of natural Ophiocordyceps sinensis specimens. Regions of interest from
high as well as single-copy number genes were successfully amplified from the
extracted DNA samples. The DNA samples obtained by this method can be
stored at )20C for over 1 year.
Conclusions: The method was effective, easy and fast and allowed batch DNA
extraction from multiple fungal isolates.
Significance and Impact of Study: Use of the thermolysis method will allow
researchers to obtain DNA from fungi quickly for use in molecular assays. This
method requires only minute quantities of starting material and is suitable for
diverse fungal species.

steps: the breaking of cell walls, and the extraction and

Introduction
As a very large group of eukaryotic organisms, the fungi
include an enormous diversity of taxa with varied ecolo-
gies, life cycle strategies and morphologies ranging from
single-celled aquatic chytrids to large mushrooms. Fungi
perform an essential role in the decomposition of
organic matter and are relevant to many human activi-
ties such as antibiotic, toxin and food production.
Although the classification and identification of fungi is
traditionally based on morphological characteristics,
recently some gene sequences (e.g., parts of the nuclear
ribosomal DNA locus) have been extensively used as
essential phylogenetic and taxonomic tools (Hibbett et al.
2007). Gene sequencing typically requires PCR, and the
initial and essential step for the amplification of a target
gene is the extraction of genomic DNA. Extraction of
fungal genomic DNA has generally involved two major
purification of genomic DNA. The genomic DNA is usu-
ally extracted with CTAB (cetyl trimethyl ammonium bro-
mide) extraction buffer (Doyle and Doyle 1987) and then
purified through phenol ⁄ chloroform extraction and iso-
propanol or ethanol precipitation (Ashktorab and Cohen
1992). Various methods have been used to break down
cell walls. In the most commonly used method, mycelia
are ground using liquid nitrogen or glass rods (Lee et al.
1988; Wu et al. 2001). In addition, researchers have also
used dry ice (Griffin et al. 2002), glass or magnetic beads
(Faggi et al. 2005), enzyme digestion (Li et al. 2002), ben-
zyl chloride (Xue et al. 2006), microwave exposure (Good-
win and Lee 1993) and combinations of different methods
(Zhang et al. 2008a). Although these techniques generally
provide DNA of satisfactory quantity and quality, most of
these techniques are tedious and time consuming and
involve the use of hazardous chemicals.

ª 2010 The Authors

114 Journal compilation ª 2010 The Society for Applied Microbiology, Letters in Applied Microbiology 51 (2010) 114–118
Y.J. Zhang et al.
In recent years, studies on fungal communities and
fungal diversity increased rapidly (Dighton et al. 2005).
These studies often require the identification of a large
number of fungal species and strains, and such identifica-
tion currently requires extraction of genomic DNA fol-
lowed by PCR amplification. Because the established
techniques for extracting genomic DNA from fungi are
time consuming, they are not suitable for use with a large
number of samples, and a simpler and faster method is
needed. The current article describes a simple ‘thermolysis
method’ for extracting fungal genomic DNA. The effi-
ciency of this method was examined by amplifying
nrDNA ITS regions of a large number of strains recov-
ered from natural Ophiocordyceps sinensis specimens. The
storage stability and target gene applicability of the geno-
mic DNA extracted with this method was also investi-
gated.
Materials and methods
Isolation and storage of fungal strains
Natural O. sinensis specimens (a type of traditional Chi-
nese medicine) were collected from Tibet and Sichuan in
China during 2003 and 2005. One strain of Hirsutella
sinensis (the anamorph of O. sinensis) (Liu et al. 1989)
and another 572 fungal strains (more than 92 species, 35
genera of three phyla) isolated from various sections
(stromata, sclerotia and external mycelial vela) of natural
O. sinensis specimens (Wang et al. 2006) were used in this
study. All strains were stored in 15% glycerol in an ultra-
low temperature refrigerator ()80C).
DNA extraction by the thermolysis method
All the fungal strains were removed from storage and
placed on PDA plates at 25C with the exception of
H. sinensis (at 20C). Once a strain had formed a colony,
a sterile toothpick was used to transfer a small amount of
mycelia or yeast cells from the colony into 100 ll of pure
water in a 1Æ5-ml microcentrifuge tube. The mixture was
vortexed thoroughly and then centrifuged at 8000–
10 000 g for 1 min. After carefully discarding the super-
natant using a pipette tip, 100 ll of lysis solution was
added to the microcentrifuge tube. The mixture was
finally incubated at 85C in a water bath for 20–30 min.
The crude extract contained genomic DNA and was
stored at )20C until use.
The ingredients of the lysis solution, which referred to
the breaking buffer in the manual of the EasySelectTM
Pichia Expression Kit (Invitrogen, USA), contained
50 mmol l)1 sodium phosphate at pH 7Æ4, 1 mmol l)1
EDTA and 5% glycerol. Before use, the lysis solution
ª 2010 The Authors
Journal compilation ª 2010 The Society for Applied Microbiology, Letters in Applied Microbiology 51 (2010) 114–118
Fungal DNA isolation by thermolysis
was autoclaved at 121C for 20 min and then stored at
4C.
DNA extraction by the CTAB method
As a comparison to the thermolysis method, genomic
DNA was extracted with the established CTAB method
(Lee et al. 1988; Wu et al. 2001). Briefly, cell walls of fun-
gal mycelia were broken down by grinding with glass rods
or in the presence of liquid nitrogen. The CTAB extrac-
tion buffer was then added, and after incubation at 65C,
purification with phenol:chloroform:isoamyl alcohol
(25 : 24 : 1) and precipitation with isopropanol were con-
ducted. Finally, the DNA was dissolved in 50 ll of pure
water. The quality of DNA samples extracted by the
CTAB method and the thermolysis method was compared
by amplifying the full-length nrDNA ITS region of H. sin-
ensis. The time required for extracting DNA with both
methods was recorded and compared using ten randomly
selected fungal strains.
Amplification of nrDNA ITS regions
Each PCR contained 2 ll of 10 · PCR buffer, 1Æ2 ll of
dNTP mixture (2Æ5 mmol l)1 each), 0Æ8 ll of deioned
formamide, 0Æ4 ll of MgCl2 (25 mmol l)1), 0Æ8 ll of each
primer (10 lmol l)1), 0Æ2 ll of Taq DNA polymerase
(5 U ll)1) and 1 ll of genomic DNA in a total volume of
20 ll. The primer pairs for amplifying ITS1 regions
were ITS1 (5¢-TCCGTAGGTGAACCTGCGG-3¢) and ITS2
(5¢-GCTGCGTTCTTCATCGATGC-3¢); those for full-
length ITS regions were ITS1 and ITS4 (5¢-TCCTCCG-
CTTATTGATATGC-3¢). A PCR consisted of an initial
denaturing step of 5 min at 94C followed by 35–40
cycles (50 s at 94C, 50 s at 54C and 50 s at 72C) fin-
ished by a final extension step at 72C for 10 min. PCR
products were resolved by electrophoresis through 1Æ2%
agarose gels in TAE (2 mmol l)1 EDTA, 80 mmol l)1
Tris-acetate, pH 8Æ0) and were visualized by staining with
GoldView (SBS Genetech, China).
Applicability and stability of the genomic DNA extracted
with the thermolysis method
The genomic DNA of H. sinensis extracted with the therm-
olysis method was used to amplify single-copy csp1 (prim-
ers CSP1-ZF ⁄ TSPa2) and MAT1-2-1 (primers MAT1-
2F1 ⁄ MAT1-2R2) genes as reported previously (Zhang et al.
2008b, 2009). With ten randomly selected fungal strains,
the stability of the DNA extracted with the thermolysis
method was tested by amplifying full-length ITS regions
with primers ITS1 ⁄ ITS4 after the DNA was stored for
1 year at )20C.
115
Fungal DNA isolation by thermolysis
L1 L2 M M M
(a) (c) (d)
2000
1000
750
500
250
100
M
(b)
Results
The thermolysis method described in this study was com-
pared with the traditional CTAB method that uses glass
rods or liquid nitrogen to break down fungal cell walls.
Good PCR products were obtained with the H. sinensis
DNA samples extracted by those two methods (Fig. 1a),
indicating that the quality of genomic DNA extracted by
both methods was comparable. To determine whether the
thermolysis method could be used to extract genomic
DNA from many different genera, the new method was
applied to various fungal strains. With this method, we
extracted the genomic DNA of the 572 fungal strains
(including ascomycetes, basidiomycetes and zygomycetes)
that were isolated from natural O. sinensis specimens. It is
not necessary to detect the genomic DNA by electropho-
resis because no visible band of the genomic DNA is
expected on agarose gels. They were used directly for
PCR amplification of nrDNA ITS1 regions with primers
ITS1 and ITS2. Without any dilution of genomic DNA
solutions, good amplification was obtained for all strains
within the genera of Mortierella, Trichocladium, Epicoc-
cum, Mucor, Trichoderma, Tricladium, Gliocladium, Toly-
pocladium, Acremonium, Beauveria and yeasts; and for
most strains within the genera of Penicillium, Paecilomy-
ces, Gymnoascus, Fusarium, Cladosporium and Neonectria
(Fig. 1b). Initial amplification, however, produced no
band or only a weak band for most strains within the
genera of Cylindrocarpon, Aspergillus, Chrysosporium, Zale-
rion, Tumularia, Cadophora, Fimetariella, Leohumicola
and Stilbella. In this instance, using a 1 ⁄ 25th-1 ⁄ 100th
dilution of the genomic DNA as a template corrected the
problem (data not shown). The failure of the initial
116 Journal compilation ª 2010 The Society for Applied Microbiology, Letters in Applied Microbiology 51 (2010) 114–118
Y.J. Zhang et al.
Figure 1 Electropherograms of PCR products
amplified with DNA samples extracted with
the thermolysis method or the established
method. (a) Comparison of DNA samples
extracted with the established CTAB method
(L1) and the thermolysis method (L2) by ampl-
ifying the full-length nrDNA ITS regions
(c. 550 bp) of Hirsutella sinensis. Markers (M)
are shown in bps. (b) Partial results for the
amplification of the nrDNA ITS1 regions
(c. 250 bp) with the DNA isolated with the
thermolysis method. (c) Amplification of the
single-copy MAT1-2-1 (left, c. 1000 bp) and
csp1 (right, c. 800 bp) genes of H. sinensis.
(d) Amplification of the full-length nrDNA ITS
regions with the DNA stored for over 1 year
at )20C.
amplification could have been caused by the presence of
PCR inhibitors in the original DNA solution. Successful
amplification was achieved with 85% of the 572 strains
using an undiluted DNA extract, but this was increased
to 100% when those that failed were diluted (data not
shown). All the amplicons were used successfully for
SSCP (single-strand conformation polymorphism) analy-
ses, and full-length ITS regions were then amplified and
sequenced for strains showing different SSCP patterns
(Zhang et al. unpublished).
The genomic DNA extracted with this simple method
is of high amplification efficiency not only for high-copy
genes (e.g., nrDNA ITS) but also for low- or single-copy
genes, such as the single-copy csp1 (one cuticle-degrading
serine protease gene) and MAT1-2-1 (one mating-type
gene) genes of H. sinensis (Fig. 1c) (15, 16).
The genomic DNA extracted with the traditional CTAB
method can be preserved for several years at )20C
(Porebski et al. 1997). We re-examined the DNA that had
been extracted with the thermolysis method and then
stored at )20C for 1 year. Good results were obtained
with PCR amplification (Fig. 1d), indicating that the
DNA extracted with the thermolysis method is stable for
at least 1 year.
Discussion
A simple and rapid method for extracting fungal genomic
DNA was proposed in this study. Compared to tradi-
tional methods, the thermolysis method has significant
advantages. First, the thermolysis method does not
involve the mechanical breaking of fungal cell walls or
DNA purification using phenol ⁄ chloroform, and the time
ª 2010 The Authors
Y.J. Zhang et al. Fungal DNA isolation by thermolysis

Table 1 Times required for three methods of extracting fungal genomic DNA

Duration for different stages (min)

Methods No. of samples treated Break ⁄ wash Incubation Purification Precipitation Total

CTAB (liquid nitrogen) One 5 30–60 35 25 95–125

Ten 50 30–60 50–60 30 165–200
CTAB (glass rods) One 3–5 30–60 35 25 93–125
Ten 30–50 30–60 50–60 30 145–200
Thermolysis One 2 20–30 – – 22–32
Ten 20 20–30 – – 40–50

The thermolysis method was compared with established CTAB methods based on liquid nitrogen or glass rods. The process for extracting genomic
DNA was divided into four stages: breaking of cell walls (for the CTAB method using liquid nitrogen or glass rods) or washing of fungal biomass
(for the thermolysis method), incubation to release DNA into buffer solutions, purification using phenol ⁄ chloroform and precipitation using isopro-
panol or ethanol. The time for each stage is estimated according to our experience. Dash (–) means the lack of a stage.

required for DNA extraction is therefore reduced greatly
(Table 1); with this method, our laboratory can process
up to 100 fungal samples in a day. Second, the thermoly-
sis method does not require liquid nitrogen or an ultra-
low temperature centrifuge, which may be unavailable to
some laboratories; the method only requires a standard
centrifuge and a water bath. It follows that this method is
practicable for a general microbial laboratory and is easily
learned by normal investigators. Third, the thermolysis
method does not use toxic chemicals (e.g., phenol or
chloroform) and so, it is safe for operators and does not
require disposal of harmful wastes. Fourth, the thermoly-
sis method requires only a small quantity of fungal bio-
mass (as little as 0Æ01 g) and so, strains do not have to be
cultivated for a long time, which is especially valuable for
slow-growing fungi, such as H. sinensis, and for the
screening of a large number of transformants in a short
time during a study of fungal genetics. Finally, contami-
nant DNA can cause significant problems with PCR
(Kwok and Higuchi 1989), but the thermolysis method
reduces the chance of contamination because it omits
many of the surface contacts (e.g., contact between DNA
and mortar, pestle, spatula and other equipment) that
occur with the traditional methods.
The thermolysis method also has limitations. Because
the method includes no purification procedures, PCR may
be inhibited by certain chemicals released from the cells of
some species ⁄ strains. These PCR inhibitors may vary with
fungal species, media used for cultivation and growth sta-
tus (Min et al. 1995; Paterson 2007, 2008). Additionally,
this method is not recommended for extracting DNA
from fungal tissues (e.g., stromata and sclerotia of O. sin-
ensis) although weak amplification can be occasionally
obtained if the genomic DNA is diluted (data not shown).
Cell walls of those fungal tissues are more difficult to
break with this method than those of mycelia, and their
DNA solutions are usually accompanied by pigments and
other chemical inhibitors of PCR amplification.
Another fast and efficient method for DNA extraction
is based on the use of microwave radiation (Tendulkar
et al. 2003). Although the quality and quantity of DNA
obtained with the microwave radiation method was
sufficient for PCR analysis and dot blot hybridization,
that method has only been used for Magnaporthe grisea;
whether the method can be applied to other fungal taxa
without protocol variations is unknown. In contrast,
the thermolysis method described here has been suc-
cessfully applied to fungal species belonging to various
taxa.
Acknowledgements
This study was supported by grants from the National
Plans of Sciences and Technology (Grants No.
2007BAI32B04), the National Outstanding Youth
Foundation (30625001), the National 863 Plan of China
(2007AA021506) and the cooperative project between
Guangdong Province and Chinese Academy of Sciences
(2009B091300015). The Authors are grateful to Ms Man-
hong Sun, Dr Bingda Sun and Dr Guozhu Zhao for
their original work in isolating and ⁄ or characterizing
fungal strains. We acknowledge Mr Wenfeng Gong for
his work in cultivating and storing the fungal strains.
The authors also thank Prof Bruce Jaffee (the University
of California at Davis) for serving as presubmission
reviewers and for his valuable comments and sugges-
tions.
References
Ashktorab, H. and Cohen, R.J. (1992) Facile isolation of geno-
mic DNA from filamentous fungi. BioTechniques 13, 198–
200.
Dighton, J., White, J.F. and Oudemans, P. (2005) The Fungal
Community: Its Organization and Role in the Ecosystem.
Boca Raton, FL: CRC Press.

ª 2010 The Authors

Journal compilation ª 2010 The Society for Applied Microbiology, Letters in Applied Microbiology 51 (2010) 114–118 117
 Fungal DNA isolation by thermolysis
Doyle, J.J. and Doyle, J.L. (1987) A rapid DNA isolation pro-
cedure for small quantities of fresh leaf tissue. Phytochem
Bull 19, 11–15.
Faggi, E., Pini, G. and Campisi, E. (2005) Use of magnetic
beads to extract fungal DNA. Mycoses 48, 3–7.
Goodwin, D.C. and Lee, S.B. (1993) Rapid, microwave
mini-prep of total genomic DNA from fungi, plants,
protists and animals for PCR. BioTechniques 15, 438–
444.
Griffin, D.W., Kellogg, C.A., Peak, K.K. and Shinn, E.A. (2002)
A rapid and efficient assay for extracting DNA from fungi.
Lett Appl Microbiol 34, 210–214.
Hibbett, D.S., Binder, M., Bischoff, J.F., Blackwell, M., Can-
non, P.F., Eriksson, O.E., Huhndorf, S., James, T. et al.
(2007) A higher-level phylogenetic classification of the
Fungi. Mycol Res 111, 509–547.
Kwok, S. and Higuchi, R. (1989) Avoiding false positives with
PCR. Nature 339, 237–238.
Lee, S.B., Milgroom, M.G. and Taylor, J.W. (1988) A rapid,
high yield mini-prep method for isolation of total genomic
DNA from fungi. Fungal Genet Newsl 35, 23–24.
Li, S.L., Zhou, B., Yang, L.Y., Li, Z.Y., Zhang, Q. and Chen,
Y.W. (2002) An improved method for extracting fungal
DNA. J Yunnan Univ 24, 471–472.
Liu, X.J., Guo, Y.L., Yu, Y.X. and Zeng, W. (1989) Isolation
and identification of the anamorphic state of Cordyceps
sinensis (Berk.) Sacc. Acta Mycol Sin 8, 35–40.
Min, J., Arganoza, M.T., Ohrnberger, J., Xu, C. and Akins,
R.A. (1995) Alternative methods of preparing whole-cell
DNA from fungi for dot-blot, restriction analysis, and
colony filter hybridization. Anal Biochem 225, 94–100.
Paterson, R.R.M. (2007) Internal amplification controls have
not been employed in fungal PCR hence potential false
negative results. J Appl Microbiol 102, 1–10.
118 Journal compilation ª 2010 The Society for Applied Microbiology, Letters in Applied Microbiology 51 (2010) 114–118
View publication stats
Y.J. Zhang et al.
Paterson, R.R.M. (2008) Fungal enzyme inhibitors as pharma-
ceuticals, toxins, and scourge of PCR. Curr Enzyme Inhib
4, 46–59.
Porebski, S., Bailey, L. and Baum, B. (1997) Modification of a
CTAB DNA extraction protocol for plants containing high
polysaccharide and polyphenol components. Plant Mol Biol
Rep 15, 8–15.
Tendulkar, S.R., Gupta, A. and Chattoo, B.B. (2003) A simple
protocol for isolation of fungal DNA. Biotechnol Lett 25,
1941–1944.
Wang, M., Sun, M.H., Zhang, Y.J., Luo, Z. and Zhuo, G.
(2006) Preliminary studies on microflora of Cordyceps
sinensis in Tibet. Edible fungi China 25(Suppl.), 6–8.
Wu, Z.H., Wang, T.H., Huang, W. and Qu, Y.B. (2001) A
simplified method for chromosome DNA preparation from
filamentous Fungi. Mycosystema 20, 575–577.
Xue, S.J., Yue, T.L., Guan, J., Yuan, Y.H. and Gao, Z.P. (2006)
An improved method for extracting fungal DNA. Food Res
Develop 27, 39–40.
Zhang, Y.H., Wei, D.S., Xing, L.J. and Li, M.C. (2008a) A
modified method for isolating DNA from fungus. Microbi-
ology China 35, 466–469.
Zhang, Y.J., Liu, X.Z. and Wang, M. (2008b) Cloning, expres-
sion, and characterization of two novel cuticle-degrading
serine proteases from the entomopathogenic fungus Cordy-
ceps sinensis. Res Microbiol 159, 462–469.
Zhang, Y.J., Xu, L.L., Zhang, S., Liu, X.Z., An, Z.Q., Wang, M.
and Guo, Y.L. (2009) Genetic diversity of Ophiocordyceps
sinensis, a medicinal fungus endemic to the Tibetan
Plateau: Implications for its evolution and conservation.
BMC Evol Biol 9, 290.
ª 2010 The Authors