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Optimization of DNA isolation from legume nodules

2007; Oxford University Press; Volume: 45; Issue: 1 Linguagem: Inglês

10.1111/j.1472-765x.2007.02149.x

1472-765X

Tatiana Krasova Wade, Marc Neyra,

Peanut Plant Research Studies

Letters in Applied MicrobiologyVolume 45, Issue 1 p. 95-99 Free Access Optimization of DNA isolation from legume nodules T. Krasova-Wade, T. Krasova-Wade Laboratoire Commun de Microbiologie Centre de Recherche de Bel Air, Dakar, SenegalSearch for more papers by this authorM. Neyra, M. Neyra Laboratoire Commun de Microbiologie Centre de Recherche de Bel Air, Dakar, SenegalSearch for more papers by this author T. Krasova-Wade, T. Krasova-Wade Laboratoire Commun de Microbiologie Centre de Recherche de Bel Air, Dakar, SenegalSearch for more papers by this authorM. Neyra, M. Neyra Laboratoire Commun de Microbiologie Centre de Recherche de Bel Air, Dakar, SenegalSearch for more papers by this author First published: 19 April 2007 https://doi.org/10.1111/j.1472-765X.2007.02149.xCitations: 14 Tatiana Krasova-Wade, IRD, UR 040, Laboratoire Commun de Microbiologie IRD/UCAD/ISRA, Centre de Recherche de Bel Air, B.P. 1386, C.P. 18524 Dakar, Senegal.E-mail: tania.wade@ird.sn Present addresses Tatiana Krasova-Wade, Laboratoire des Symbioses Tropicales et Méditerranéennes (LSTM), 34398 Montpellier Cedex 5, France Mark Neyra, IRD, UMR 113 IRD/CIRAD/AGRO-M/UM2, USC INRA 1242, Laboratoire des Symbioses Tropicales et Méditerranéennes (LSTM), Campus de Baillarguet, TA10/J, 34398 Montpellier Cedex 5, France. AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Abstract Aims: The aim of this study was to optimize DNA extraction from legume nodules to obtain large amounts of high-quality genomic DNA. Methods and Results: Nodules of different legume species were used. Varied concentrations of guanidine thiocyanate (from 6 mol l−1 to 0·05 mmol l−1), a component of DNAzol, were tested. The quality of DNA extract was determined by PCR–RFLP. The best results were obtained with 0·5 mmol l−1 guanidine thiocyanate, which resulted in greater DNA yield than with higher and lower concentrations or with DNAzol. Conclusion: The procedure using 0·5 mmol l−1 guanidine thiocyanate yields the highest DNA amount when compared with previously described protocols and offers a reliable method to isolate DNA from nodules of different origins. Significance and Impact of the Study: Irrespective of nodule origin, DNA yield was increased significantly, by two (e.g., Vigna nodules) to seven (Acacia auricoliformis nodules) times. In addition, the proposed procedure's costs are lower than those using the DNAzol. Introduction Isolation protocols of genomic DNA from different samples including soil, vegetable matter, bacteria, mushrooms and blood are widely used. Previously, protocols for DNA isolation from Frankia nodules were proposed (Baker and Mullin 1994; Rouvier et al. 1996). One of these protocols was slightly modified and adapted to legume nodules (Andréet al. 2003, 2005; Krasova-Wade et al. 2003) and has been successfully carried out, making it possible to study diversity of rhizobia in nodules without strain isolation, in particular by PCR–RFLP (restriction fragment length polymorphism) or direct sequencing of 16S-23S rDNA IGS. However, the procedure remains toxic, time-consuming and expensive for large serial analysis. Moreover, different centrifugation steps cause a progressive elimination of vegetal material and, therefore, reduction of DNA yield. A rapid, non-toxic and easy methodology of DNA isolation from liquid and solid samples with DNAzol (Chomczynski et al. 1997) has been proposed. The present study proposes an adaptation of the use of guanidine thiocyanate, composed of DNAzol reagent, for isolation and purification of total DNA from legume nodules. Several authors have obtained high-yield DNA with variable concentrations of guanidine salts (Lippke et al. 1987; Pitcher et al. 1989; Huang et al. 2000). This report presents various modifications of the previously reported protocols allowing improved DNA yield and an assessment of the extraction cost. In addition, the usefulness of the proposed DNA preparation for nodules of different origins is presented. Materials and methods Nodule collection Nodules were collected from plants of different legume species: cowpea (Vigna unguiculata L. Walp.), Acacia mangium, Acacia auriculiformis, A cacia crassicarpa, Acacia senegal, Leucaena leucocephala, and Gliricidia sepium species. The plants were inoculated with rhizobia in nursery conditions in non-sterile soil at the experimental station in Laboratoire Commun de Microbiologie (IRD/UCAD/ISRA) in Dakar, Senegal. Harvested nodules were dried at 45°C for 48 h and then stored at room temperature. DNA isolation procedure Nodules were re-hydrated in sterile distilled water and surface sterilized by immersion in 3·3% w/v Ca(OCl)2 for 3 min, rinsed in sterile water, then in absolute ethanol for 2–3 min, followed by rinsing in sterile water. They were crushed together in TES/sucrose (20 mmol l−1 Tris–HCl, pH 8·0, 50 mmol l−1 EDTA disodium, pH 8·0, 50 mmol l−1 NaCl, 8% w/v sucrose) buffer by using plastic pestles (MERCK Eurolab, 60678.01, Strasbourg, France) sterilized in absolute ethanol. We propose the following protocol: each nodule (3·3–4·0 mg) was crushed in 100 μl of TES/sucrose buffer with a plastic pestle sterilized in absolute ethanol in a 1·5-ml Microcentrifuge tube (Rex 2000). 10 μl of lysozyme (20 mg μl−1) was added to the crushed nodule and the homogenate was mixed by vortexing for 20 s and then incubated at 37°C for 15 min for lysis. 250 μl of GES reagent (0·5 mmol l−1 guanidine thiocyanate, 0·1 mol l−1 EDTA disodium, pH 8·0, 1% w/v N-Lauroylsarcosine sodium salt) were added to the lysed material. It was mixed by vortexing for 20 s, and then incubated at 65°C for 15 min. At this step, the mixture can be stored for 3 days at 4°C. It was centrifuged at 19 000 g for 15 min at 4°C, and then the supernatant was transferred to a new microtube. The total DNA was precipitated from the supernatant by adding 0·5 vol (180 μl) 95% EtOH at room temperature, mixed by inversion and stored at room temperature for 3 min. The DNA was recovered by centrifugation at 4°C at 19 000 g for 15 min. The supernatant was eliminated. The DNA pellet was washed with 0·5 ml 95% EtOH at room temperature, and then centrifuged at 4°C at 19 000 g for 15 min. At this step, the DNA in EtOH can be stored for 2 months at 4°C. The supernatant was discarded and the pellet was rinsed with 0·5 ml 95% EtOH. After centrifugation at the same conditions, EtOH was removed and the DNA pellet was air-dried. It was resuspended and dissolved in a volume (50–250 μl) of 8 mmol l−1 NaOH for complete dissolution. Before PCR, the pH of DNA extract was adjusted to 7·5 with 8 μl of 0·1 mol l−1 HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) per 50 μl DNA extract. The purity and the quantity of DNA yield were estimated by spectrophotometry by using a Model Ultrospec 3000 UV/Visible Spectrophotometer (Pharmacia Biotech, Cambridge, UK) in the range 200 to 340 nm and compared to these methods. The DNA preparation was stable when stored at −20°C. All centrifugations were done in a 3 K15 Model Centrifuge (SIGMA). Statistic analyses Data of DNA yields were analyzed with Superanova software (Abacus Concepts Inc., Cary, NC, USA). Fisher's PLSD method was used to separate the means at P < 0·05 level of significance when comparing different treatments on cowpea nodules. The Newman-Keuls method of STATITCF software (version 5, copyright-1987–88–91, Institut Technique des Céréales et des Fourrages, Paris, France) was used to separate the means at P < 0·05 level of significance for data comparison on tree nodules. Molecular analysis Polymerase chain reaction (PCR) was performed using Ready-to-Go Taq DNA Polymerase (Pharmacia Biotech) and RFLP was carried out under previously described conditions (Krasova-Wade et al. 2003). 16S-23S primers designed from conserved regions of Frankia sp. rrn, FGPS1490-72 (located at positions 1490-1510 of the 16S rDNA, 5′-TGCGGCTGGATCCCCTCCTT-3′) (Normand et al. 1996) and FGPL132-38 (located at positions 132-114 of the 23S rDNA, 5′-CCGGGTTTCCCCATTCGG-3′) (Normand et al. 1992) were used for PCR amplification of 16S-23S rDNA IGS region. Results and discussion Initially, the optimization of DNA extraction was carried out on cowpea nodules. DNA was extracted following three methods: (i) modified DNAzol protocol (Rex 2000; Ligozzi and Fontana 2003), (ii) CTAB/PVPP (hexadecyltrimethylammonium bromide/polyvinylpolypyrrolidone) with phenol : chloroform : isoamyl alcohol purification (Krasova-Wade et al. 2003) and (iii) the GES protocol described above. To facilitate comparison between treatments, 36 nodules harvested on cowpea plants inoculated with the same strain were pooled together. The homogenized material was distributed into three Eppendorf tubes for each treatment. At the first time, we did carry out the test on the DNAzol method (i). It did not produce a different DNA yield than the CTAB/PVPP method (ii), but incurred a higher cost. We then tested the less expensive GES reagent (Pitcher et al. 1989) following the DNAzol procedure, but we varied guanidine thiocyanate concentrations. Different incubation times (30, 15, 10 and 5 min) and concentrations (0·05 mmol l−1 to 6 mol l−1) of guanidine thiocyanate in the GES method were tested at 65°C. The highest DNA yield was obtained with 15 min incubation (4·8 mg g−1 dry matter compared with 4·0 and 4·5 mg g−1) and with 0·5 mmol l−1 guanidine thiocyanate (Table 1). The DNA yield increased significantly at 2·7 times when compared with the CTAB/PVPP method, and at 2·27 times when compared with the DNAzol method. The GES procedure permitted the recovery of pure nucleic acids (without proteins or RNA) with an absorbance ratio A260/280 of 1·75 and molecular weight bigger than 10 kb (Fig. 1a) (Lippke et al. 1987; Chomczynski et al. 1997), ready for PCR–RFLP analysis of rhizobia DNA without any additional purification (Fig. 1b,c). Table 1. DNA yield and purity obtained with different methods on cowpea nodules Treatment Nodule dry weight (g)* Protein purity A260/A280† DNA quantity (mg g−1 dry matter)†,‡ CTAB/PVPP§ 0·0036 1·63 1·17abc DNAzol/lys/65°C 0·0036 1·5 1·42bc GES (6 mol l−1)** 0·0036 1·71 1·06ab GES (5 mol l−1)** 0·0036 1·58 1·43bc GES (1 mol l−1)** 0·0036 1·7 1·65bc GES (0·1 mol l−1)** 0·0036 1·78 1·77bc GES (0·005 mol l−1)** 0·0036 1·68 2·1c GES (0·001 mol l−1)** 0·0036 1·75 2·0bc GES (0·0005 M)†† 0·0036 1·75 3·22d GES (0·0001 mol l−1)** 0·0036 1·66 2·0c GES (0·00005 mol l−1)** 0·0036 1·58 1·78bc Without guanidine thiocyanate** 0·0036 2·55 0·4a *Mean weight of one dry nodule determined as the ratio between the weight of all of nodules in crushed mixture and nodule number. †Purity of nucleic acid samples in the presence of protein and RNA contaminations. Results are given as the mean of three replicates. ‡Values in the column that are followed by different letters are significantly different at P < 0·05 by the Fisher's PLSD test. §Results obtained with CTAB/PVPP method (Krasova-Wade et al. 2003). **Results obtained with different concentrations of guanidine thiocyanate (given in parenthesis) of GES reagent (this study). ††Results obtained with DNAzol method (Lippke et al. 1987; Rex 2000). Figure 1Open in figure viewerPowerPoint Electrophoresis of (a) total DNA isolated from cowpea nodules by GES method, (b) 16S-23S IGS rDNA PCR product and (c) 16S-23S IGS rDNA PCR product digested with HaeIII endonuclease. Lanes 1–5, individual cowpea nodules. Gel (a): 0·8% agarose; lane M, KiloBase DNA Marker (Amersham). Gel (b): 1·0% agarose; lane M, KiloBase DNA Marker (Amersham); lane C, amplification control without DNA. Gel (c): 2·5% Metaphor gel, lane M, 100 Base-Pair Ladder (Pharmacia Biotech). Arrows indicate the DNA extract. We applied the optimized GES method on dried nodules of different tree legumes. Data were compared with those obtained from the CTAB/PVPP method (Table 2). The results showed that, irrespective of nodule origin, the GES method provides three (e. g., Leucaena nodules) to seven (Acacia auricoliformis nodules) times higher DNA yield than the CTAB/PVPP method, with similar A260/280 ratios being acceptable for PCR–RFLP and suitable for other molecular applications (Fig. 2). The size of amplified DNA corresponded to the expected size of the 16S-23S IGS region of rhizobia (Normand et al. 1996). Table 2. DNA yield obtained on tree nodules by GES protocol Tree species Nodule dry weight (g)* Protein purity A260/A280† DNA quantity (mg g−1 dry matter)† GES CTAB/PVPP GES CTAB/PVPP Acacia mangium 0·0025 1·5 1·67 1·67a 0·39b Acacia auricoliformis 0·005 1·3 1·55 2·06a 0·28b Acacia crassicarpa 0·005 1·6 1·63 0·76a 0·20b Acacia senegal 0·005 1·4 1·53 0·83a 0·21b Leucaena leucocephala 0·005 1·7 1·45 0·87a 0·26b Gliricidia septum 0·0025 1·4 nd 0·93 nd nd, not determined *Mean dry weight of one nodule determined as a ratio between the weight of all of crushed nodules in mixture and nodule number. †Purity of nucleic acid samples in the presence of protein and RNA contaminations. Results are given as the mean of three replicates for both DNA extraction protocols, with GES or with CTAB/PVPP. Values in the row that are followed by different letters are significantly different at P < 0·05 by the Newman–Keuls's test. Figure 2Open in figure viewerPowerPoint PCR electrophoresis of 16S-23S IGS rDNA of total DNA isolated from nodules of different tree legumes. Lanes M, KiloBase DNA Marker (Amersham); 1–7, individual nodules of Acacia auriculiformis, A. crassicarpa, A. senegal, A. mangium, A. anguistissima, Leucaena leucocephala and Gliricidia sepium. The goal of this study was to examine the effect of different guanidine thiocyanate concentrations on the effectiveness of DNA extraction, as it has never been tested by the existing methods. Our results showed that increasing DNA yield could be obtained by decreasing guanidine thiocyanate concentration from 6 mol l−1 to 0·5 mmol l−1. At 0·5 mmol l−1 guanidine thiocyanate concentration, the maximum amount of DNA was recovered. This procedure offers a reproductive method to isolate DNA from nodules of different legume species. Additionally, it is safer than the method (ii) (phenol-, chloroform- and CTAB-free) and rapid (1 h 20 min per nodule). 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