Martín& Winka 2000_lichenologits_amplificacion_pcr_beads

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 protocols 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 precipitated 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). Polysaccharides 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

*The subscript indicate the part of th e specimen used (t: thallus; a: apothecium). ^Preliminary study.

Xanthoria resendei

Teloschistes villosus%

AF101286 AF101281 AF101282 AF098411 AF098410 AF101274 AF101275 AF101276 AF101277 AF101278 AF101279 AF101280 AF098409 AF098405 AF098406 AF098408 AF098407 AF101285 AF101283 AF101284

Accesion number 23 vii 1996 20 iii 1997 20 iii 1997 26 iv 1997 28 iv 1997 23 iv 1997 18 v 1997 18 v 1997 18 v 1997 18 v 1997 29 iii 1997 30 ix 1996 23 iv 1997 18 i 1997 28 iv 1997 28 iii 1997 28 iii 1997 20 iii 1997 20 iii 1997 03 iv 1993

Collection date 27 v 1997 05 v 1997 05 v 1997 05 v 1997 05 v 1997 28 iv 1997 27 v 1997 27 v 1997 27 v 1997 27 v 1997 29 iii 1997 28 iv 1997 28 iv 1997 16 iv 1997 05 v 1997 16 iv 1997 20 v 1997 05 v 1997 05 v 1997 05 v 1997

Extraction date

g TS

»

rt O

n

3

3, CtDt Ct D, CtDt

ft

Dta

n »

A la B ta C,l D t a

Ca Da C, D, At B't Ct' D t

Da Dt Dt Da Da

Ca Da Ca D a

CtDt C,D,

D,

Extraction method*

lih

Protoparmelia sp. Teloschistes chrysophthalmus Teloschistes lacunosus\

Protoparmelia psarophana

Diploschistes ocellatus Diploschistes ocellatus var. almeriensis Lecanora pulicaris Protoparmelia montagnei

BCC-Lich BCC-Lich BCC-Lich BCC-Lich BCC-Lich BCC-Lich BCC-Lich BCC-Lich BCC-Lich BCC-Lich BCC-Lich BCC-Lich BCC-Lich BCC-Lich BCC-Lich BCC-Lich BCC-Lich BCC-Lich BCC-Lich BCC-Lich

Buellia rivas-maninezii Caloplaca gloriac

13204 13177 13177 13207 13208 13258 13178 13178 13178 13179 13180 12261 13258 13173 13205 13174 13203 13175 13176 13259

Voucher number

Species

TABLE 1. Specimens used for DNA extraction

to

o o o

DNA ext Win

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 1 2

3

4

5

193

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 amplification from both the mycobiont and the phycobiont, the presence of more than one mycobiont or multiple rDNA types. With the primer pair ITS1FITS4, 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 amplification 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

THE LICHENOLOGIST

194

1 2

3

4

5

Vol. 32

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. 2. 3. 4. 5. 6. 7. 8.

9. 10. 11. 12. 13.

Place sample in a sterile 1-5-ml Eppendorf tube. Grind sample with a micropestle for 30 s. Add 100 ul of buffer and grind 30 s-1 min. Add 200 ul of buffer and grind 30 s-1 min. Add 500 ul buffer and grind 30 s-1 min. Add 100 ul of 3% SDS. Mix by shaking three times. Incubate at 55°C overnight. 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. Repeat step 8, three or four times, until no more proteins remain in the interface. 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). Centrifuge at 14 000 rpm for 15 min. Clean pellet with 70% ethanol. Centrifuge at 6 000 rpm for 5 min. 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 polysaccharides 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. REFERENCES

Armaleo, D. & Clerc, P. (1991) Lichen chimeras: DNA analysis suggests that one fungus forms two morphotypes. Experimental Mycology 15: 1-10. Armaleo, D. & Clerc, P. (1995) A rapid and inexpensive method for the purification of DNA from lichens and their symbionts. Lichenologist 27: 207-213. Boom, R., Sol, C. J. A., Salimans, M. M. M., Jansen, C. L., Wertheim-van Dillen, P. M. E. & Noordaa van der, J. (1990) Rapid and simple method for purification of nucleic acids. Journal of Clinical Microbiology 28: 495-503. Bruns, T. D., Fogel, R. & Taylor, J. W. (1990) Amplification and sequencing of DNA from fungal herbarium specimens. Mycologia 82: 175-184. Crespo, A., Bridge, P. D. & Hawksworth, D. L. (1997) Amplification of fungal rDNA-ITS regions from non-fertile specimens of the lichen-forming genus. Parmelia. Lichenologist 29: 275-282. Cubero, O., Crespo, A., Fatehi, J. & Bridge, P. D. (1999) DNA extraction and PCR amplification method suitable for fresh, herbarium-stored, lichenized and other fungi. Plant Systematics and Evolution 216: 243-249. Gardes, M. & Bruns, T. (1993) ITS primers with enhanced specificity for basidiomycetesapplication to the-identification of mycorrhizae and rusts. Molecular Ecology 2: 113-118. Grube, M., DePriest, P. T., Gargas, A. & Hafellner, J. (1995) DNA isolation from lichen ascomata. Mycological Research 99: 1321-1324. Landvik, S., Shailer, N. F. J. & Eriksson, O. (1996) SSU rDNA sequence support for a close relationship between the Elaphomycetales and the Eurotiales and Onygenales. Mycoscience 37: 237-241. Lee, S. B. & Taylor, J. W. (1990) Isolation of DNA from fungal mycelia and single spores. In PCR Protocols: a Guide to Methods and Applications (M. A. Innis, D. H. Gelfand, J. J. Sninsky & T. J. White, eds): 282-287. San Diego: Academic Press. Lee, S. B., Milgroom, M. G. & Taylor, J. W. (1988) A rapid, high-yield mini-prep method for isolation of total genomic DNA from fungi. Fungal Genetics Newsletter 35: 23-24. White, T. J., Bruns, T., Lee, S. B. & Taylor, J. W. (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In PCR Protocols: a Guide to Methods and Applications (M. A. Innis, D. H. Gelfand, J. J. Shinsky & T. J. White, eds): 315-322. San Diego: Academic Press. Whiting, M. F., Carpenter, J. C , Wheeler, Q. D. & Wheeler, W. C. (1997) The Strepsiptera problem: Phylogeny of the holometabolous insect orders inferred from 18S and 28S ribosomal DNA sequences and morphology. Systematic Biology 46: 1-68. Winka, K., Ahlberg, C. & Eriksson, O. E. (1998) Are there lichenized Ostropales? Lichenologist 30: 455-462.

Accepted for publication 18 November 1999