Lichenologist 32(2): 189-196 (2000)

doi: 10.1006/lich. 1999.0254

Available online at http://www.idealibrary.com on IDEKL

ALTERNATIVE METHODS OF EXTRACTING AND

AMPLIFYING DNA FROM LICHENS

Maria P. MARTIN* and Katarina WINKAJ

Abstract: We have investigated whether DNA extraction protocols designed

specifically for fungi and/or lichens perform better on lichens than do corresponding
protocols designed for plants and insects. Two different PCR-amplification proto-
cols were used to evaluate the quality of the DNA extracted with each method. The
DNA extractions with highest quality were obtained with the protocols designed for
insects and plants, and the most successful amplifications were obtained with
Ready-To-Go PCR Beads. This indicates that fungal or lichen specific protocols
might not be necessary for successful extraction of high quality DNA from lichens.
(C 2000 The British Lichen Society

Introduction
A number of different methods for the extraction and amplification of DNA
from lichens and fungi have been published (e.g. Armaleo & Clerc 1991;
1995; Bruns et al. 1990; Crespo et al. 1997; Cubero et al. 1999; Grube et al.
1995; Landvik et al. 1996; Lee et al. 1988; Lee & Taylor 1990). In general, the
authors of these protocols argue that fungi and lichens require specially
designed protocols, because these organisms are rich in polysaccharides, and
may also contain polyphenols and/or tannins. These compounds need to be
removed from the extracted DNA since they can be inhibitory to the further
enzymatic analysis of the DNA. The protocols mentioned above can be
separated into two groups, which differ mainly in (1) the chaotropic agents
included in the lysis buffers, (2) the purification steps to remove proteins,
polyphenols and/or polysaccharides and (3) the DNA precipitation.
In Armaleo & Clerc (1995), Bruns et al. (1990), Crespo et al. (1997),
Cubero et al. (1999) Lee & Taylor (1990), and Lee et al. (1988), the secondary
products are extracted with phenol:chloroform or chloroform after incubation
in hot lysis buffers containing a detergent such as sodium dodecyl sulphate
(SDS) or cetyl-trimethyl ammonium bromide (CTAB). The DNA is precipi-
tated with isopropanol. In Armaleo & Clerc (1991), Grube et al. (1995), and
Landvik et al. (1996), the chaotropic salt guanidine thiocyanate denatures the
proteins and the DNA is precipitated onto silica particles (glassmilk). Polysac-
charides are removed by washing the DNA-binding glassmilk pellet with
ethanol.

*Departamento de Biologia Vegetal (Botanica), Facultad de Biologia, Avda. Diagonal 645,

08028-Barcelona, Spain.
^Department of Ecology and Environmental Science, Umea University, SE-90187 Umea,
Sweden.
0024-2829/00/020189 + 08 $35.00/0 © 2000 The British Lichen Society
190 THE LICHENOLOGIST Vol.32
We have experienced varying results when using these protocols, and
because of this, we have tried also other methods, designed for extracting
DNA from other organisms. In this work, we evaluate if specific methods
really are necessary when working with lichens. We have used two protocols
designed for these organisms: Lee & Taylor (1990) and Landvik et al. (1996),
one protocol designed for extraction of DNA from insects (Whiting et al.
1997), and one commercial kit for extraction of plant DNA (Nucleon
Phytopure Plant DNA Extraction Kit). There are many more methods and
commercial kits available, but these four protocols were chosen because they
have been used frequently by us, and because they represent the two 'groups'
of protocols described above. The plant DNA extraction kit, like the protocols
by Lee & Taylor (1990) and Whiting et al. (1997), use SDS buffers and
chloroform and/or phenol to lyse the cells and remove proteins, whereas
Landvik et al. (1996) use a guanidine-thiocyanate buffer and glassmilk. All
protocols require minimal amounts of material and are easy to set up. To
evaluate the quality of the extracted DNA, we also tested two different
polymerase chain reaction (PCR) amplification protocols.
The lichens included in this study belong to the ascomycete families
Parmeliaceae, Physciaceae and Teloschistaceae (Lecanorales) and Thelotremata-
ceae. (Graphidales). Some of them are very closely related and systematic
studies of this group are in progress (Martin, unpublished).

Materials and Methods

Herbarium specimens of the lichens included in this study (Table 1) were visually examined, and
only portions of the specimens (less than 0-01 g) that appeared to be in good condition were used
for the extractions. Specimens of the same species are differentiated by their Genbank accession
numbers. Two studies were performed, a preliminary and an extensive study. In the preliminary
study, all four DNA extraction protocols, and two kinds of PCR protocols were tested on two
species: Teloschistes lacunosus (AF098405) and T. villosus (AF098408). From the results of
this study, we then chose the protocols with the best results for an extensive study of all 20 taxa
(Table 1).
DNA extraction
Each one of the four extraction protocols were prepared in duplicate.
Protocol A. The protocols by Lee & Taylor (1990) and Bruns et al. (1990) differ only in
microcentrifugation times in the DNA-precipitation step, so we consider them as the same here.
The lysis buffer contained 50 mM ethylenediaminetetraacetic acid (EDTA), 50 mM Tris-HCl
(pH 7-2), 3% SDS and 1% p mercaptoethanol (added just before use). After incubating the
sample at 65°C for 1 h, the DNA was cleaned once with phenol: chloroform (1:1) and a second
time with phenol:chlorofom:isoamyl alcohol (25:24:1). The DNA was precipitated with 3M
sodium acetate (NaOAc) (pH 5-2) and isopropanol.
Protocol B. The Landvik et al. (1996) protocol uses sonication to disrupt the cell walls of the
fungal material, but this step was excluded. The guanidine lysis buffer was prepared as described
in Boom et al. (1990): 120 g guanidinium thiocyanate (GuSCN) was dissolved in 100 ml of 0-1
M Tris-HCl (pH 6-4), and 22 ml 0-2 M EDTA-solution adjusted with NaOH to pH 8'0, and
2-6 g of Triton X-100 added. The fungal material was incubated in the guanidine buffer for
30 min at 56°C. Then the DNA was precipitated with glassmilk and 8-2 M Nal, and the pellet was
washed with cold 70% ethanol.
Protocol C. Nucleon Phytopure Plant DNA Extraction Kit (Scotlab Biosciences, Scotland) was
used. The cells were lysed in a buffer containing SDS. Chloroform was then added along with a
 to
o
o
o

TABLE 1. Specimens used for DNA extraction

Species Voucher number Accesion number Collection date Extraction date Extraction method*
DNA ext

Buellia rivas-maninezii BCC-Lich 13204 AF101286 23 vii 1996 27 v 1997 D,

Caloplaca gloriac BCC-Lich 13177 AF101281 20 iii 1997 05 v 1997 CtDt 3
BCC-Lich 13177 AF101282 20 iii 1997 05 v 1997 C,D, n
Diploschistes ocellatus BCC-Lich 13207 AF098411 26 iv 1997 05 v 1997 rt
O
Diploschistes ocellatus var. almeriensis BCC-Lich 13208 AF098410 28 iv 1997 05 v 1997 Ca Da »
Lecanora pulicaris BCC-Lich 13258 AF101274 23 iv 1997 28 iv 1997 Ca D a
Protoparmelia montagnei BCC-Lich 13178 AF101275 18 v 1997 27 v 1997 Da
BCC-Lich 13178 AF101276 18 v 1997 27 v 1997 Dt g
BCC-Lich 13178 AF101277 18 v 1997 27 v 1997 Dt TS
Protoparmelia psarophana
lih

BCC-Lich 13179 AF101278 18 v 1997 27 v 1997 Da

BCC-Lich 13180 AF101279 29 iii 1997 29 iii 1997 Da n
Protoparmelia sp. BCC-Lich 12261 AF101280 30 ix 1996 28 iv 1997 Ca Da »
Teloschistes chrysophthalmus BCC-Lich 13258 AF098409 23 iv 1997 28 iv 1997 C, D,
Teloschistes lacunosus\ BCC-Lich 13173 AF098405 18 i 1997 16 iv 1997 At B't Ct' D t
BCC-Lich 13205 AF098406 28 iv 1997 05 v 1997
Teloschistes villosus% BCC-Lich 13174 AF098408 28 iii 1997 16 iv 1997 A la B ta C,l D t a ft
BCC-Lich 13203 AF098407 28 iii 1997 20 v 1997 Dta 3,
Xanthoria resendei BCC-Lich 13175 AF101285 20 iii 1997 05 v 1997 CtDt
BCC-Lich 13176 AF101283 20 iii 1997 05 v 1997 Ct D,
BCC-Lich 13259 AF101284 03 iv 1993 05 v 1997 CtDt
Win

*The subscript indicate the part of th e specimen used (t: thallus; a: apothecium).
^Preliminary study.
192 THE LICHENOLOGIST Vol. 32
Nucleon Phytopure proprietary resin. The specially modified resin particles contain free boric acid
[-B(OH)2] groups that covalently binds polysaccharides and thereby removes them from the
sample. The DNA was precipitated with isopropanol.
Protocol D. For the Whiting et al. (1997) protocol, the lysis buffer was made up of 50 mM
EDTA, 50 mM Tris-HCl and 3% SDS. After incubation at 55°C for 12 h, the DNA was cleaned
with phenol:choloroform:isoamyl alcohol (25:24:1), the process was repeated four or five times,
followed by precipitation of DNA in cold absolute ethanol (2 h 30 min at -20°C).
All pellets obtained with the four protocols were resuspended in autoclaved Milli-Q water.
Extraction methods A and D are quite similar. But, in protocol A, 1 % [S-mercaptoethanol is added
to the extraction buffer to inhibit the polyphenol oxidization process, and in D the incubation time
in lysis buffer is 12 h instead of 1 h.

PCR amplification
The primer pairs ITS1F/ITS4, ITS1/ITS4, ITS5/ITS4, ITS5/ITS2 and ITS3/ITS4 were used
to amplify the internal transcribed spacer 1 (ITS1), the 5-8S rRNA gene, and the internal
transcribed spacer 2 (ITS2) of the ribosomal RNA gene cluster, as described by White et al.
(1990) and Gardes & Bruns (1993). Amplification reactions were done using two protocols. First,
a standard procedure where PCR reactions were done in a total reaction volume of 20 ul
containing 1-5 ul of 50% glycerol (cocktail with 1-5 ul of Milli-Q water was also tested), 2 |il PCR
reaction buffer (10 x PCR Buffer II) provided by Perkin Elmer, 200 mM deoxynucleotides
(dNTP) (Pharmacia), 1-5 mM MgCl2, 10 pmol of each primer and 0-5 units of AmpliTaq" Gold
DNA Polymerase (Perkin-Elmer).
The second protocol was Ready-To-GoR PCR Beads (Amersham-Pharmacia Biotech). The
beads provide the reagents for the PCR reactions in a convenient ambient-temperature-stable
bead. The beads have been optimized for PCR reactions and contain buffer, nucleotides and Taq
DNA Polymerase. The only reagents that have to be added are the template DNA and the specific
primers, thus the number of pipetting steps are reduced. This yields better reproducibility and
minimizes the risk of contamination. Individual reactions to a final volume of 25 ul were carried
out with two different primer concentrations (5-10 pmol ul ').
Negative controls, without DNA template, were prepared in each series of amplifications in
order to detect possible contaminations in reagents or reaction buffers. To verify that the cocktails
were functional, a positive control {Bipolaris urochloae) was also included in every series of
amplifications. Before the PCR cycling was initiated, the samples were denatured at 94°C for
12 min when using AmpliTaq R Gold Polymerase, and for 5 min when using PCR Beads. In both
protocols, the cycling parameters were: 5 cycles of denaturation at 94°C for 30 s, annealing at
55°C for 30 s, and extension at 72°C for 1 min, followed by 33 cycles of denaturation at 94°C for
30 s, annealing at 48°C for 30 s and extension at 72°C for 1 min, with a final extension at 72°C
for 10 min. The PCR amplifications were performed in a Perkin-Elmer Cetus DNA Thermal
cycler (GeneAmp 2400).
Results from the amplifications were monitored by electrophoresis of 5-|il aliquots in a 1%
Seakem Agarose gel (FMC Bioproducts) (Figs 1-4). When little product was obtained (weak
bands in the gels), re-amplifications were made: (a) cut the fragment from the gel, (b) put the
piece in a 1-5-ml Eppendorf tube, (c) place the tube in the freezer for 10 min, (d) place it in the
microwave for 5 min, (e) add 200 ul Tris-EDTA (TE), (f) incubate 5 min at 90°C, (g) dilute 1:10
and 1:100 and use as template, (h) run the amplification reaction with PCR Beads and the same
cycling parameters and primers as in the first amplification.

Results and Discussion

Preliminary study
The total working time required for each extraction protocol was: (A) 2 h
30 min of which 1 h is incubation time; (B) 2 h 40 min including 1 h of
incubation; (C) 2h 30 min including 30 min of incubation time; (D) 16 h
including 12 h of incubation time and 2 h 30 min for DNA precipitation.
2000 DNA extraction and amplification—Martin & Winka 193

1 2 3 4 5 6

600 bp

FIG. 1. PCR amplifications of ITS rDNA using PCR Beads and primer pair ITS1F/ITS4. Lanes
1,3,5 extraction DNA protocol C. Lanes 2,4,6 extraction protocol D. Lanes 1-2 Caloplaca
gloriae (GLOCAL-1-1). Lanes 3-4 C. gloriae (GLOCAL-1-2). Lanes 5-6 Xanthoria resendei
(RESXAN-1).

For both amplification protocols, and both species, more than one fragment
was obtained when performing PCR reactions with the primer pairs ITS5/
ITS4, ITS1/ITS4, ITS5/ITS2 and ITS3/ITS4. As mentioned in Crespo et al.
(1997), using universal primers, multiple PCR products may indicate ampli-
fication from both the mycobiont and the phycobiont, the presence of more
than one mycobiont or multiple rDNA types. With the primer pair ITS1F-
ITS4, amplifications were successful, but only when using PCR Beads and
DNA from extraction protocols C and D. No significant differences were
observed concerning the quality of the PCR products obtained from these two
protocols. The successful amplification obtained when using DNA from
protocol D as template suggests that the overnight incubation at 55°C breaks
the cell and organelle membranes of the mycobiont more efficiently than did
the inclusion of 1 % p"-mercaptoethanol in protocol A.
Extraction protocols A, B and D have almost the same cost in money and
work time; D took longest time because of the overnight incubation. The
quality of the DNA was without doubt best in D, because we achieved
stronger amplifications. The commercial protocol C is most expensive, and
most time consuming, but the quality of the DNA was equal to that obtained
with protocol D. For the PCR amplifications the PCR Beads are by far the
most expensive, but the results were much better than with the standard PCR
protocol. The combination of DNA from extraction protocol D, and ampli-
fication with PCR Beads, produced good products not only from the lichens
used in this study, but also from fungal basidiomata and cultures (Martin &
Garcia, unpublished).

Extensive study
Our conclusion from the preliminary study was that extraction protocols C
and D worked best and that amplifications were most successful using the
primer pair ITS1F/ITS4 and PCR-Beads. In the extensive study these
protocols were applied to most of the remaining 17 taxa (Table 1).
Both extraction methods C and D produced a visible DNA precipitate
and good PCR products were obtained (Figs 1 & 2) with the following
exceptions: (a) in Teloschistes lacunosus (AF098406) no product was obtained
when using DNA from extraction C as template, although good amplifications
were obtained from extraction D (Fig. 3); (b) in Lecanora pulicaris, no
194 THE LICHENOLOGIST Vol. 32

1 2 3 4 5 6

— 600 bp

FIG. 2. PCR amplifications of ITS rDNA using PCR Beads and primer pair ITS1F/ITS4;
extraction DNA protocol D. Lane 1 Protoparmeliapsarophana (PSAPRO-1). Lane 2 P. psarophana
(PSAPRO-2). Lane 3 P. montagnei (MONPRO-M). Lane 4 P. montagnei (MONPRO-1-2).
Lane 5 P. montagnei (MONPRO-1-3). Lane 6 Buelliarivas-martinezii(RIVBUL-1).

1 2 3 4 5 6

— 600 bp

FIG. 3. PCR amplifications of ITS rDNA using PCR Beads and primer pair ITS1F/ITS4. Lanes
1,3,5 extraction DNA protocol C. Lanes 2,4,6 extraction DNA protocol D. Lanes 1-2 Teloschistes
lacunosus (LACTEL-2). Lanes 3-4 Diploschistes ocellatus var. almeriensis (OCEDIP-1). Lanes 5-6
D. ocellatus (OCEDIP-2).

—1018 bp

FIG. 4. PCR amplifications of ITS rDNA using PCR Beads and primer pair ITS5/ITS4 from
Lecanora pulicaris, Lane 1 extraction DNA protocol C. Lane 2 extraction DNA protocol D.

product was obtained when using primers ITS1F/ITS4. However, the primer
pair ITS5/ITS4 resulted in sufficient product. The fragments were stronger
when DNA from extraction D was used (Fig. 4).
2000 DNA extraction and amplification—Martin & Winka 195
TABLE 2. Modified DNA extraction protocol from Whiting et al. (1997)

1. Place sample in a sterile 1-5-ml Eppendorf tube.

2. Grind sample with a micropestle for 30 s.
3. Add 100 ul of buffer and grind 30 s-1 min.
4. Add 200 ul of buffer and grind 30 s-1 min.
5. Add 500 ul buffer and grind 30 s-1 min.
6. Add 100 ul of 3% SDS. Mix by shaking three times.
7. Incubate at 55°C overnight.
8. Add 500 ul of phenolxhloroform: isoamyl alcohol (25:24:1). Shake
gently, but continuously, for 5 min. Centrifuge at 14 000 rpm for 5 min.
Transfer upper phase to a new 1-5-ml Eppendorf tube, be sure to
eliminate proteins from the interface.
9. Repeat step 8, three or four times, until no more proteins remain in the
interface.
10. Precipitate DNA with 750 ul absolute ethanol and keep the tubes at
- 20°C for 2 h 30 min (or 30 min at - 80°C or overnight at 4°C).
11. Centrifuge at 14 000 rpm for 15 min.
12. Clean pellet with 70% ethanol. Centrifuge at 6 000 rpm for 5 min.
13. Dry pellet for 30 min at room temperature. Resuspend the DNA with
200 ul of Milli-Q sterile water.

In general, extraction method D gave stronger products than method C. As

mentioned in Grube et al. (1995), many of the DNA isolation protocols
published up to now allow the quick extraction of DNA but do not remove
many proteins and polysaccharides that interfere with molecular techniques. In
method C the Nucleon Phytopure resin proved effective in removing polysac-
charides from the sample. The four orfivepurification steps in method D also
produced clean and usable DNA. Moreover, the good quality of the isolated
DNA is maintained through the months, as was demonstrated when the DNA
extractions of Diploschistes ocellatus var. almeriensis and D. ocellatus were used to
amplify and sequence the 18S rRNA gene, 7 months later (Winka et al. 1998).
Both strands of the PCR products were sequenced with an Applied
Biosystems 377 Automatic Sequencer (Foster City, CA, USA) using the
AmpliTaq DNA Polymerase FS Dye Terminator Cycle Sequencing kit
(Perkin Elmer). The sequences obtained were of good quality.
According to our results, the most cost- and time-effective combination
seems to be extraction protocol D and amplification with PCR Beads. The
DNA is of high purity and the amount is sufficient for more that 200 PCR
reactions. In Table 2 we give the complete extraction protocol of Whiting et al.
(1997) including our modifications. In conclusion, it seems that it is not
necessary to use fungal or lichen specific protocols to successfully extract
DNA from lichens, since the protocols that proved to be most effective in our
study were the ones developed for plants (Nucleon PhytoPure) and insects
(Whiting et al. 1997). These protocols both use lysis buffers with SDS, and
chloroform and/or phenol to remove proteins, but the increased incubation
time and repeated phenol/chloroform purifications in Whiting et al. (1997),
and the specially modified resin particles in the Nucleon Phytopure Plant
DNA Extraction Kit, are probably the reasons why these protocols were more
efficient in removing polysaccharides and proteins from the extracted DNA.
196 THE LICHENOLOGIST Vol. 32
There are, of course, additional commercial kits available that may be even
better than the one we have tested. K. Winka has used the DNeasy Plant Mini
Kit (QIAGEN), which seems comparable to the Nucleon kit, both in time,
cost and quality of results. M. Martin has also tried EZNA Fungal Kit
(OmegaBiotech) with very good results.
We are grateful to Ove Eriksson (Umea University, Sweden) for providing laboratory facilities,
and to Mary Berbee (University of British Columbia, Canada; visiting researcher at Umea
University during 1997) for supplying the positive control and for her advice during the laboratory
work. We also thank Manuel Casares (University of Granada), Xavier Llimona and Antonio
Gomez (University of Barcelona) for providing the collections using in this study, and Marian
Glenn (Seton Hall University, New Jersey, USA) for correcting the English. This study was
supported by a grant to Ove E.Eriksson from the Swedish Natural Science Research Council, and
a travel grant from the British Mycological Society to M. P. Martin.

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Accepted for publication 18 November 1999