Development of new procedures for the isolation of phytoplankton DNA from fixed samples Elena Bertozzini1 , Antonella Penna2 , Elisa Pierboni3 , Ian Bruce4 & Mauro Magnani5,∗ 1
Centre of Biotechnology, University of Urbino, Via Campanella 1, 61032 Fano (PU), Italy; 2 Centro Biologia Ambientale, University of Urbino, Vle Trieste 296, 61100 Pesaro (PU), Italy; 3 Istituto Zooprofilattico Sperimentale Umbria e Marche, Via Salvemini 1, 06126 Perugia (PG), Italy; 4 School of Chemical and Life Sciences, University of Greenwich, Kent ME4 4TB, U.K.; 5 Institute of Biological Chemistry “G. Fornaini”, University of Urbino, Via Saffi 2, 61029 Urbino (PU), Italy (∗ Author for correspondence; e-mail:
[email protected], phone: +39-0722-305211; fax: +39-0722-320188)
Received 17 September 2004; revised and accepted 6 February 2005
Key words: DNA extraction, fixatives, harmful algal blooms (HABs), magnetisable solid phase support (MSPS), PCR Abstract Phytoplankton samples collected for routine monitoring programmes have traditionally been preserved with fixatives before subsequent analytical procedures such as microscope-based identification, or simply to permit transport between laboratories. In recent years, to simplify identification and enumeration, the use of DNA or RNA probes coupled with the PCR assay has progressed and now represents a routine procedure for screening cultured and field samples. However, the phytoplankton cells have often still to be treated as fixed samples. The extraction of genomic DNA from fixed cultures of Alexandrium minutum cultures was compared using two new methods based on Magnetisable Solid Phase Support (MSPS) techniques with that using three commercial kits. Genomic DNA recovery and PCR amplification were observed and the results obtained from culture samples were validated using field samples. Among the DNA extraction techniques considered, the MSPS methods provided the best results. Abbreviations: DEAE, Diethylaminoethyl group; HAB, Harmful Algal Bloom; ITS, Internal transcribed spacer; MSPS, Magnetisable Solid Phase Support; PEG, polyethylene glycol Introduction Microscope-based microalgal cell identification methods are usually the standard procedures used in laboratories for the screening of cultured and natural samples. Conventional light microscopy has been extended to the use of other techniques, such as phase contrast, fluorescence microscopy and scanning electron microscopy (SEM) for the specific identification of phytoplanktonic species. The detection of toxic microalgae in field samples is often time-consuming using light and electronic microscopy and requires both experience and significant taxonomic skills (Godhe et al., 2002). The advanced nucleic acid techniques have
revolutionized the methodological approach permitting a more rapid identification of microalgae species in mixed phytoplankton samples through the application of different molecular methods such as those using antibodies, lectins, DNA and RNA probes (Anderson et al., 1999; Cho et al., 1999; Bolch, 2001). In particular, nuclear ribosomal DNA (rDNA) has been extensively used as a molecular target for the identification of HAB (Harmful Algal Bloom) species in diagnostic PCR, sandwich and whole cell hybridization assays (Scholin et al., 1999; Penna & Magnani, 1999; Guillou et al., 2002). When usinging PCR, the genomic DNA has to be extracted and purified from samples, removing potential inhibitors that often cause PCR inhibition
224 or low yields of PCR products (A. Penna, pers. comm.). The efficiency of the genomic DNA extraction step is very important for the subsequent PCR assay, especially when it is to be used in quantitative investigation on cultured or environmental samples. Amplification efficiency is guaranteed by complete cellular lysis and careful purification of target DNA (Huang et al., 2000; Mar`ın et al., 2001). Furthermore, in the sampling of long-term monitoring programmes the PCR assay must be applied to preserved natural samples. Fixatives, such as Lugol’s solution, formalin and glutaraldehyde, are used as preserving agents for the longterm storage of phytoplankton material before morphological and molecular identification, especially when a number of samples are collected during cruises or monitoring activities. In this study, two new methods for DNA extraction based on super-paramagnetic beads were developed; one method is based on the application of silica-coated magnetic beads, the other uses a different MSPS (Magnetisable Solid Phase Support), which contains agarose as support material. The agarose is derivatized with diethylaminoethyl (DEAE) group (Bruce et al., 1996; Levison et al., 1998). The silica-magnetite beads adsorb DNA under high salt conditions and release it when salt concentration decreases (Davies et al., 1998; Taylor et al., 2000). The agarose-magnetite MSPS beads bind DNA through the DEAE groups and subsequently the elution of the genomic DNA is obtained via ion competition. Both methods were applied for total DNA extraction from fresh and fixed samples using cultures of the HAB dinoflagellate A. minutum and environmental samples. A. minutum is a dinoflagellate commonly involved in HAB events in the Mediterranean Sea (Vila et al., 2004). After total DNA extraction from cultured and natural samples, the PCR amplificabilty of the genomic DNA was examined by a PCR reaction designed to amplify the target sequences of the 5.8S rDNA-ITS regions. Then, the performance of these two new methods was compared with three commercial kits presently in use: PUREGENETM DNA Purification Kit (Gentra Systems) which employs alcohol precipitation; R DNeasy plant Mini Kit (Qiagen) which utilizes a spin R column containing a siliceous resin and Dynabeads TM DNA DIRECT Universal Kit (Dynal Biotech) which also employs super-paramagnetic beads. The technological implications of the application of these methods in the isolation of PCR-ready DNA are discussed.
Materials and methods Sample collection Alexandrium minutum strain CNR-AMIA1 (provided by Dr M.G. Giacobbe, Istituto per l’Ambiente Marino Costiero, CNR, Messina, Italy) was grown in batch culture of f/2- enriched seawater medium (Guillard, 1975) for 28 days at 18 ◦ C. Light was provided by cool-white fluorescent tubes at a photon flux of 100 µmol m−2 s−1 on a 14:10 LD cycle. Samples containing 250,000 A. minutum cells (cell density was determined by enumeration using a hemacytometer (Neubauer, Hausser Scientific, Horsham, PA)) were used for total DNA extraction. The first batch of samples was fixed with 0.2% (v/v) Lugol’s solution, the second batch was fixed with 4% (v/v) neutral formalin solution and the last batch was centrifuged at 2000 × g for 15 min; the supernatants were discarded and pellets conserved at −20 ◦ C until the day of the extraction to serve as unfixed control. Formalinfixed samples and Lugol’s solution-fixed samples were stored at 4 ◦ C until the day before the extraction. Successively, samples were centrifuged at 2000 × g for 15 min and the pellets were washed twice with 1 mL of sterile seawater. We chose to freeze the pellets of the fixed cells as well, to simulate both the same conditions for both fresh and fixed samples and beacause this step could facilitate cell lysis. 50 mL of seawater were collected during an A. taylori bloom in proximity of the Vulcano Island (Aeolian Island, Sicily, Italy) in June 2003. This field sample, containing 1.5 × 106 cells (29 × 106 cells L−1 ), was immediately fixed by addition of 0.2% (v/v) Lugol’s solution, divided into five aliquots, and stored at 4 ◦ C. The samples were then centrifuged at 2000 × g for 15 min, washed twice with sterile seawater and frozen at −20 ◦ C. DNA extraction Silica-magnetite beads were produced as previously described by Taylor et al. (2000). For genomic DNA extraction, cell pellets were thawed at room temperature and resuspended in 75 µL of 50 mM Tris HCl pH 8.0 containing 50 mM EDTA and 0.5% (v/v) Triton X100. 1.5 µL of 10 mg/mL RNase A and 5 µL of 20 m/mL proteinase K were added to the lysates and the mixtures were then incubated at 50 ◦ C for 10 min. 50 µL of 4 M guanidine hypochlorite (pH 5.5) were added, the samples were vortexed and incubated at 65 ◦ C for
225 10 min. The tubes were vortexed once more and the lysates were added to 50 µL of silica-magnetite beads previously washed four times with 1 mL of distilled H2 O (dH2 O). A 100-µL volume of 4 M NaCl containing 20% (w/v) PEG was added to each sample and the mixtures were gently agitated for 10 min at room temperature. The silica-magnetite beads-DNA-complex suspensions were immobilized using a magnetic stand and the supernatants discarded. Then, 200 µL of 2 M NaCl were added, the suspensions immobilized and the supernatants discarded. The silica-magnetite beads were washed twice with 50% (v/v) ethanol for 2 min, the suspensions were again immobilized and the supernatants discarded. The adsorbed DNA was eluted by addition of 50 µL dH2 O and the resulting mixture was incubated at 65 ◦ C for 10 min; the suspension was then immobilized and the supernatant conserved in a new 1.5 mL tube. DEAE-agarose-MSPS beads were produced as previously reported (Bruce et al., 1996). Frozen cell pellets were thawed at room temperature and resuspended in 75 µL 50 mM Tris-HCl buffer (pH 8.0) containing 0.05 M EDTA, 0.5% (v/v) Triton X100 and 200 µg mL−1 RNase A. These suspensions were incubated with 5 µL of a 20 mg mL−1 aqueous solution of proteinase K (SigmaAldrich Corporation, St.Louis, USA) at 37 ◦ C for 30 min. The mixtures were clarified by vortexing with 25 µL of 3 M guanidine hypochlorite (pH 5.5) and incubated at 50 ◦ C for 30 min. Each lysate was diluted adding a 1.2 mL volume of sterile dH2 O and this mixture was added to 150 µL of DEAE-agarose magnetic beads previously washed as follows: three washes with 1 mL of dH2 O, one wash with STET Buffer (50 mM Tris-HCl pH 8.0, 0.001 M EDTA, 0.01 M NaCl, 1% (v/v) Triton X100), and a final wash with 1 mL dH2 O before the addition of the lysate. The DEAE-agarose beads-DNA complex suspensions were gently mixed for 5 min at room temperature, the beads were immobilized and the supernatants were discarded. The beads were washed by resuspension in 400 µL 0.01 M Tris-HCl pH 8.0 buffer containing 0.4 M NaCl and 0.001 M EDTA. After immobilization of the beads, the supernatants were discarded and the genomic DNA desorbed by incubation with 200 µL 0.05 M arginine-free base solution in 1.0 M NaCl at 65 ◦ C for 5 min. After immobilization on the magnetic stand, the supernatants were transferred to a fresh 1.5 mL tube. Additional genomic DNA was isolated by
one further desorption step as described above and the supernatants pooled. The DNA obtained through these two elution steps was precipitated with pure ethanol, washed with 70% ethanol and resuspended in 20 µL of dH2 O (Sambrook et al., 1989). DNA extractions using PUREGENETM DNA Purification Kit (Gentra Systems, Minneapolis, Minnesota, R USA); DNeasy plant Mini Kit (Qiagen, Valencia, R USA) and Dynabeads DNA DIRECTTM Universal Kit (Dynal Biotech, ASA, Norway) were performed on thawed pellets according to the standard protocols suggested by the manufacturers. Quantification of genomic DNA Genomic DNA concentrations were assessed fluorometrically using the DNA Quantitation Kit Fluorescence assay (Sigma-Aldrich Corporation, St.Louis, USA) and evaluating the fluorescence of bisbenzimide (Hoechst 33258) which binds double-stranded DNA. The bisbenzimide fluorescence emission at 460 nm, when excited with 360 nm light, increases significantly in the presence of DNA (Portugal & Waring, 1988). Fluorescence was quantified using a spectrofluorophotometer RF-5301PC (Shimadzu, Kyoto, Japan). PCR amplification Total genomic DNA extracted from each sample was tested in the PCR reactions. The 5.8S rDNA and flanking internal transcribed spacers (ITS1 region and ITS2 region) were amplified using ITSA and ITSB primers (Adachi et al., 1994). These primers were synthesized by SIGMA-Genosys (Sigma-Aldrich Corporation, St.Louis, USA). Amplifications were performed in an Applied Biosystems DNA Thermo Cycler 2400 (Foster City, USA). Reaction tubes contained a 25 µL mixture of: 200 µM of each of dNTP; 15 pmol of each primer; 2 mM MgCl2 ; 1× Reaction Buffer (Finnzymes Oy; Espoo, Finland); 0.4 U Taq Polymerase (Finnzymes Oy; Espoo, Finland) and a variable amount of template DNA. To test the sensitivity of the PCR assay, we performed the amplification on 10, 1 and 0.1 ng total genomic DNA extract from fresh, formalin and Lugol fixed samples. PCR conditions were as follows: an initial denaturation step of 10 min at 95 ◦ C, 30 cycles of 30 at 94 ◦ C, 30 at 55 ◦ C and 1 at 72 ◦ C, and a final extension step of 10 at 72 ◦ C. 10 µL of PCR products were resolved on a 1.8 % (w/v) agarose, 0.5× TBE buffer gel and were visualized
226 Table 1. Total genomic DNA extracted from 250,000 A. minutum CNR-AMIA4 cells obtained by the five DNA extraction methods. Silicamagnetite beads
Genomic DNA purification Kit GENTRA SYSTEMS
R DNeasy Plant Mini Kit QIAGEN
R Dynabeads DNA DIRECTTM Universal DYNAL
183 ± 75
204 ± 97
320 ± 168
189 ± 58
Lugol’s solution-fixed samples
1064 ± 43
120 ± 57
221 ± 106
317 ± 192
150 ± 49
Neutral Formalin-fixed samples
680 ± 43
150 ± 41
188 ± 76
352 ± 101
12 ± 2
Unfixed samples
523 ± 151
DEAEagarose MSPS
Note. Presented values are mean ± standard deviation of three extractions. Yields are expressed in nanograms.
by standard ethidium bromide staining under UV light (Sambrook et al., 1989).
Results The five extraction methods yielded different amounts of total genomic DNA extract (Table 1); the values presented in Table 1 were determined fluorometrically and show substantial agreement with gel image analysis results (data not shown). The analysis of the data was carried out comparing the DNA recovery of the five methods investigated and the recovery within the same method, to evaluate to what extent the presence of fixative substances influenced DNA extraction. The variances of the results were compared using the Anova test. When the genomic DNA was extracted using Gentra, Qiagen or DEAE-agarose-MSPS methods, yields of total DNA extracted were not influenced by the fixing process ( p < 0.01). When genomic DNA was extracted using silica-magnetite beads or R Dynabeads , the Anova test carried out on the amount of extracted DNA under each condition gave p values higher than 0.01 for both methods, clearly indicating that the presence of fixative substances strongly influenced DNA extraction. When applying the Dynal method, the recovery of total genomic DNA significantly decreased when the starting material was fixed with formalin (Table 1). In contrast, when genomic DNA extractions were performed on Lugol-fixed samples and using silica-magnetite beads, DNA recovery was higher. Among the five methods, when focusing attention on fixed samples a simple comparison of genomic DNA yields permitted the classification of extraction efficiency. Silica-magnetite beads gave the highest yields from both Lugol and formalin-fixed samples. To assess the quality of the recovered DNA, the various genomic DNAs obtained by the five extraction methods were used as template for a PCR reaction on the 5.8S-ITS regions, in order to evaluate the level of
contaminants present in the final eluates. PCR products were visualized on a 1.8% (w/v) agarose gel. In all the PCR reactions, the PCR products obtained from the A. minutum strains were approximately 610 bp long. The DNA extracted with silica-magnetite beads from fixed samples shows good amplificability. PCR products were clearly visible on agarose gel, while the genomic DNA extracted from fresh samples was poorly amplifiable (Figure 1A). Genomic DNA obtained by DEAE-agarose-MSPS gave the best results in the PCR amplification assay. In fact, PCR products were clearly visible under each condition with no differences between fixed and unfixed samples (Figure 1B). Genomic DNA extracted with the Gentra Systems method was amplifiable under each condition. However, when the DNA template was derived from fixed cells, the intensity of PCR bands was lower and at a template concentration of 0.1 ng, the PCR reaction yielded bands at the detection limit of the resolving gel (Figure 1C). When R was the genomic DNA extracted with Dynabeads used as a template in the PCR reactions, PCR products were always visualized on the gel, with the exception of those derived from formalin-fixed samples which produced band at the resolving limit of agarose gel in electrophoretic analysis (Figure 1D). Genomic DNA obtained by the Qiagen method was amplified and all PCR products were visualized on agarose gel except for the PCR fragment in lane 1 (Figure 1E) which was not amplified. The five different methods for DNA extraction were also applied to seawater samples containing a monospecific bloom of A. taylori. Genomic DNA purified by each procedure was quantified spectrofluoremetrically. Silica-magnetite beads and DEAE-agarose MSPS beads gave the highest yields (Figure 2). PCR was performed on 1 ng DNA template as described in the material and methods section, and 10 µl of PCR products were visualized on a 1.8% (w/v) agarose gel. Figure 3 shows that PCR amplifications were observed under each condition.
227
Figure 2. Amounts of genomic DNA extracted from a fixed field sample containing A. taylori cells using the five extraction procedures. S stand for silica-magnetite beads; D for DEAE-agaroseR R MSPS beads; Dy, stand for Dynabeads (Dynal); Q for DNeasy Plant Mini Kit (QIAGEN) and G for PuregeneTM DNA purification system (Gentra systems).
Figure 3. PCR amplified products using ITSA-ITSB primers from 1 ng of genomic DNA extracted from a field Lugol-fixed sample containing A. taylori cells. The genomic DNA was extracted with Silica-magnetite beads (lane 1); DEAE-Agarose MSPS beads (lane R 2); DNeasy Plant Mini Kit-QIAGEN- (lane 3); PuregeneTM DNA R purification system-Gentra systems (lane 4); Dynabeads -Dynal (lane5). Lane M, 50–1000 bp size standards; (lane C), positive control (plasmidic DNA of A. taylori), (lane B), blank (no DNA).
Discussion Figure 1. PCR amplified products using ITSA-ITSB primers from three different dilution of genomic DNA obtained from A. minutum CNR AMI-A4 cultured samples extracted with the five DNA extraction methods: Silica-magnetite beads (A); DEAE-Agarose-MSPS beads (B); PuregeneTM DNA purification system-Gentra systems R R (C); Dynabeads -Dynal (D); DNeasy Plant Mini Kit QIAGEN) (E); Lane M, 150–2100 bp standards, except for Figure 1E, lane M, 50–1000 bp size standard; lanes 1, 2 and 3 PCR products obtained from 10, 1 and 0.1 ng of genomic DNA extracted from unfixed samples, respectively; lanes 4, 5 and 6 PCR products obtained from 10, 1 and 0.1 ng of genomic DNA extracted from Lugol-fixed samples, respectively; lanes 7, 8 and 9 PCR products obtained from 10, 1 and 0.1 ng of genomic DNA extracted from formalin-fixed samples, respectively.
This study permitted two new genomic DNA extraction methods based on MSPS technology to be applied to cultures of A. minutum and seawater samples. Silicamagnetite beads allowed optimum yields particularly when genomic DNA extraction is performed on the fixed cultured samples (Table 1). It appears that the more chaotropic conditions facilitate the interaction between the silica shell and DNA. The amount of recovered genomic DNA was very high and the yields were coupled with adequate amplificability in the PCR reactions (Figure 1A). The genomic DNA obtained from
228 preserved samples therefore represents a valid template for the PCR reactions. Similar results were not observed when unfixed samples were investigated, the genomic DNA extracted being poorly amplifiable. A possible explanation may be the presence of PCR inhibitors, since PCR performance is improved at lower template concentration. The DEAE-agarose-MSPS method shows good results when performed on fixed samples, yields are not significantly different from those obtained with the other methods, but the amplificability of genomic DNA is very successful in the PCR assays (Figure 1B); moreover these results are confirmed on unpreserved samples. The three commercial kits considered in this study give good yields of genomic DNA both when applied to unfixed and fixed samples, with the exception of Dynal kit (Table 1), but the amplificability of the extracted genomic DNA is negatively influenced by the presence of fixative substances (Figure 1). The results from field samples validate those obtained from culture samples and confirm that, when working with fixed samples, the use of silica-magnetite or DEAE-agarose-MSPS methods guarantees high yields and valid performance in PCR reactions. In conclusion, these results show that the new methods employing silica-magnetite beads and DEAE-agarose beads represent a valid alternative for obtaining PCR-ready microalgal DNA when compared to the three commercial kits used for genomic DNA mini-prep. When a high DNA yield from preserved samples is required, the use of silica-magnetite beads is to be preferred. The DEAE-agarose method allows the highest genomic DNA PCR amplificability in both preserved and unpreserved samples. These new methods can be applied on preserved phytoplankton samples with a satisfactory recovery of genomic DNA making possible its use as template in PCR reactions since no problems due to inhibition were observed, with the additional advantage of being able to use the same sample for both microscopic and molecular investigations.
Acknowledgments We thank Dr Maria Grazia Giacobbe (Istituto Ambiente Marino Costiero, CNR, Messina, Italy) for kindly providing A. minutum CNR-AMIA1 strain. This work
was partially supported by the EU STRATEGY project contract no. EVK3-CT-2001-00046 (2001) and EU CHEMAG project G5RD-CT-2001-00534.
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