Chromoplast DNA Isolation: Plastid DNA Purification Methods from Carrot
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Highly purified DNA is required to study the physical and chemical characteristics of plastid DNA. Yet its isolation is hampered by secondary-origin substances — phenolic compounds, polysaccharides and others — together with active nucleases. It is also worth remembering that a purification method that yields clean DNA preparations from one object will not necessarily give positive results with another.
This was confirmed by our own experiments on purifying DNA from the plastids of carrot roots and leaves. Various modifications of the phenol–chloroform–detergent method, fractionation on hydroxyapatite columns, and the use of cetavlon all failed to give positive results.
The DNA preparations obtained in this way did not meet the requirements for clean native DNA. They showed contamination with proteins and polysaccharides and a low hyperchromic effect (Table 1).
The purity and native state of the preparations improved when certain stages of different methods were combined. Combining phenol–chloroform–detergent deproteinization with chromatography on hydroxyapatite allowed us to obtain plastid DNA preparations that were well purified from proteins (A260/A230 = 1.8–1.85) and polysaccharides (A260/A230 = 1.95–2.05) and that had a high level of nativity (the hyperchromic effect was in the range of 33–38 %), regardless of the plant species studied.
Table 1. Characteristics of DNA preparations from chromoplasts of red carrot roots of the Kharkivska Nantska variety, isolated using different procedures
| Procedure | A260/A230 | A260/A280 | Hyperchromic effect, % |
| V. P. Lobov et al. | 1.4 | 1.6 | 20 |
| Yu. M. Sivolap, A. N. Kotlov | 1.4 | 1.6 | 21 |
| G. E. Sulimov, A. G. Slyusarenko | 1.6 | 1.3 | 18–22 |
| R. Kolodner, K. Tewari | 1.6 | 1.8 | 20 |
| R. Britten et al. | 1.4 | 1.7 | 26 |
| G. E. Sulimova et al. | 2.0 | 1.4–1.6 | 22–25 |
| V. P. Lobov, I. A. Petrov | 1.95–2.05 | 1.8–1.85 | 33–38 |
Removing nuclear DNA impurities is a crucial step in obtaining purified plastid DNA preparations. On average, a single nucleus contains as much DNA as is present in a thousand plastids. Consequently, even unnoticed contamination of preparations of chloroplasts, chromoplasts or other plastids with nuclear fragments can distort the study of the physical and chemical characteristics of plastid DNA.
To eliminate possible nuclear DNA impurities, plastids are incubated in the presence of nucleases. R. Herrmann and co-authors showed that combining DNase and phosphodiesterase treatments completely removes nuclear DNA impurities from plastid preparations of virtually any plant. The principle of the nuclease treatment is that DNase and phosphodiesterase readily penetrate the intact nuclear envelope but do not enter intact plastids.
During nuclease treatment all nuclear DNA is destroyed, as is the DNA of organelles whose membrane envelopes are damaged, while the DNA of intact plastids remains in the native state. There is also another way of removing nuclear impurities. H. Bohnert and E. Crouse, while studying the conditions for obtaining purified chloroplast DNA preparations, found that repeated washing of plastids in a buffer containing EDTA is also effective at removing nuclei and their fragments.
In obtaining DNA preparations from the plastids of carrot roots and leaves, V. P. Lobov and I. A. Petrov used both nuclease treatment and washing of the organelles in an EDTA-containing medium. A reliable criterion of plastid DNA purity is its capacity for rapid renaturation.
Plastid DNA, characterized by low kinetic complexity compared with nuclear DNA, reassociates rapidly after denaturation, whereas denatured nuclear DNA reassociates over a long period. The carrot plastid DNA preparations obtained by V. P. Lobov and I. A. Petrov reassociated to their original absorbance at a wavelength of 260 nm within 120 minutes (Fig. 1).
This indicated the absence of nuclear contamination in the plastid DNA preparations, which made it possible to use them in studying thermal denaturation and reassociation kinetics.
Figure 1 — Restoration of the native state of non-fragmented denatured DNA: chloroplasts (a) and chromoplasts (b) of Kharkivska Nantska carrot (1); chromoplasts of Mshak carrot (2); amyloplasts of the white green-headed carrot variety (3). The dashed line indicates thermal denaturation of DNA up to a temperature of 98 °C; the solid line indicates DNA renaturation at 60 °C.
How did V. P. Lobov and I. A. Petrov isolate DNA from carrot plastids?
The method developed by V. P. Lobov and I. A. Petrov for isolating DNA was applied to the chromoplasts of red carrot roots of the Kharkivska Nantska variety, as well as to chromoplasts of the yellow Mshak carrot, amyloplasts of the white green-headed carrot, and chloroplasts from the leaves of these carrot varieties. The sequential stages are described below.
The fraction of isolated plastids was suspended in an isolation medium (0.05 M Tris, 0.5 M mannitol, 0.03 M ascorbic acid, 0.01 M EDTA, 0.1 % bovine serum albumin, pH 7.8–7.9) containing 10 mM magnesium chloride. DNase was added at a rate of 100 µg/ml, and the suspension was incubated for 30 minutes at 0 to +4 °C with constant stirring.
Snake-venom phosphodiesterase was then added at 30 µg/ml and incubation continued for a further 20 minutes. To stop the action of the nucleases, a threefold volume of alkaline EDTA solution (0.15 M NaOH, 0.1 M EDTA, pH 8.0) was added, and the plastids treated in this way were sedimented by centrifugation for 30 minutes at 2,500 rpm. The plastid pellet was lysed in a 10-fold volume of alkaline EDTA containing 3 % sodium dodecyl sulfate.
The lysate was extracted with equal volumes of water-saturated phenol (pH 8.0), then a phenol:chloroform mixture (4:1), and finally ether — twice in each case. The lower phase was then dialyzed overnight against 0.24 M sodium phosphate buffer, pH 6.8, treated for 4 hours with pronase (50 µg/ml), brought to 8 M urea and 1 M NaCl, and purified on a hydroxyapatite column. To do this, the resulting DNA solution was passed through a hydroxyapatite column previously equilibrated with a solution containing 0.18 M sodium phosphate buffer, 1 M NaCl and 8 M urea.
The column was washed with the same solution until the eluate showed no absorbance at 260 nm. During these procedures the DNA adsorbs onto the hydroxyapatite, while contaminating substances are not retained on the column. The column was then washed with 0.10 M sodium phosphate buffer to remove urea, and the DNA was eluted with 0.4 M sodium phosphate buffer.
What procedure did R. Herrmann use for DNA from daffodil organelles?
R. Herrmann applied the following procedures when isolating DNA from the organelles of the yellow daffodil. The organelles were suspended in 2 volumes of 0.3 M NaCl, 0.03 M Tris, pH 8.7 (or 0.3 M NaCl, 0.03 M sodium citrate, pH 8.0) and lysed for 10 minutes at room temperature with sodium dodecyl sulfate (5 %). The mixture was gently shaken with an equal volume of chloroform and octanol (6:1) and centrifuged for 15 minutes at 8,000 g.
The aqueous layer and the interphase were combined, gently mixed with phenol saturated with 0.03 M Tris, pH 8.7, containing 5 drops of 32 % NaOH per 100 ml of phenol, and centrifuged for 20 minutes at 10,000 g. The phenol treatment and centrifugation were repeated. The aqueous phase and interphase were extracted twice with ether and dialyzed at 2 °C against 0.4× (0.15 M NaCl, 0.015 M sodium citrate, pH 8.0) overnight. The solutions were then treated with 40 µg/ml DNase-free RNase A and 200 units/ml RNase T1 for 90 minutes at 37 °C.
After this, treatment with DNase-free pronase was carried out with the addition of 0.15 % sodium dodecyl sulfate for 4 hours at 40 °C. The enzymes were removed by phenol deproteinization, and the aqueous phase was dialyzed against the appropriate buffer and subjected to further purification in a cesium chloride density gradient. For this, the DNA solution was brought to a density of 1.710 g/cm³.
Centrifugation was performed in 5 ml tubes in a bucket rotor at 33,000 rpm for 68 hours at 20 °C. The gradient was fractionated dropwise, the DNA-containing fractions were collected, dialyzed against 0.3× (0.15 M NaCl, 0.015 M sodium citrate), and used to study physical and chemical properties.
How are circular DNA molecules isolated from daffodil chromoplasts?
For isolating circular molecules from the chromoplasts of the yellow daffodil and the tulip, the method previously applied to identify circular molecules in spinach chloroplasts was used. To 2 ml of a suspension of purified plastids was added 2 ml of a solution containing 0.79 M sucrose, 0.15 M NaCl, 0.1 M EDTA, 50 mM Tris-HCl, pH 9.0, along with sodium dodecyl sulfate (to a final concentration of 0.5 %), pronase (1 mg/ml) and sodium deoxycholate (1 %).
During incubation the mixture was stirred at 4 °C, followed by the continuous addition of five-molar sodium perchlorate to a final concentration of 1 M, and held for 30 minutes at 4 °C with 0.5 volume of chloroform:isoamyl alcohol (24:1) and 1 volume of phenol saturated with 0.15 M NaCl, 0.1 M EDTA, 50 mM Tris, pH 9.0. The mixture was centrifuged at 1,000 g for 15 minutes at 4 °C.
The aqueous phase was removed and dialyzed overnight in the cold against 0.15 M NaCl, 0.015 M sodium citrate, 0.5 mM EDTA, pH 8.0. After these operations the DNA was ready for electron-microscopic studies. An essential point in the described method is the use of pronase, which breaks down proteins and releases intact DNA molecules.
This is possible, however, only if the enzyme preparation contains no DNase impurities. J. Thompson, while studying the physical and chemical characteristics of DNases, used the method of R. Kolodner and K. Tewari — developed for the chloroplasts of higher plants — to isolate chromoplasts of the garden nasturtium.
According to this procedure the plastid fraction, after treatment with DNase and washing with EDTA, was sedimented and then resuspended in 4.8 ml of buffer A containing 0.05 M Tris, 0.002 M EDTA, pH 8.0, and pronase (200 µg/ml). To the suspension was added 1.2 ml of 10 % sodium sarkosyl in the same solution, and the mixture was incubated for 30 minutes at 37 °C in a glass centrifuge tube. Then 2 ml of buffer A containing 4 M cesium chloride was added, and the tubes were placed on ice for 90 minutes.
The mixture was centrifuged for 30 minutes at 1,200 g in the cold (4 °C), and the supernatant was transferred to nitrocellulose tubes that had been soaked overnight in 10 % sodium sarkosyl. To each centrifuge tube was added 3.2 g of cesium chloride and 0.2 ml of ethidium bromide solution (10 µg/ml). Each tube was underlaid with a 3 ml cushion containing cesium chloride (ρ = 1.74), 0.05 M Tris, 0.01 M EDTA, 200 µg/ml ethidium bromide, pH 8.0, and the tubes were centrifuged for 12–16 hours in a Spinco SW 41 rotor at 25,000 rpm.
After the centrifuge stopped, the tubes were examined under long-wave ultraviolet light (365 nm) and the fluorescing DNA bands were selected. The plastid DNA from six gradients was pooled and placed in sarkosyl-treated nitrocellulose tubes. The DNA solutions were then diluted with 5 ml of a solution containing cesium chloride (ρ = 1.57), 0.05 M Tris, 0.01 M EDTA, 200 µg/ml ethidium bromide, pH 8.0, and the DNA was re-centrifuged for 30–48 hours in a Spinco SW 41 rotor at 32,000 rpm.
Ethidium bromide was removed from the DNA-containing gradient fraction by extraction with isopentyl alcohol saturated with 0.01 M EDTA, and the DNA was dialyzed against three changes of 0.1 M NaCl, 0.05 M Tris, 0.01 M EDTA, pH 8.0 (500 ml). Using this method to isolate DNA from the chromoplasts of garden nasturtium petals allowed J. Thompson to obtain preparations consisting of 52 % circular and 48 % linear molecules.
How do you choose the right DNA isolation method?
The choice of DNA isolation method depends on the goal of the experiment. For studying thermal denaturation and reassociation kinetics, for example, where a high degree of purity is required but fragmented DNA is used, it is better to apply methods that do not require ultracentrifugation.
For isolating DNA for electron-microscopic studies and restriction analysis, centrifugation in a cesium chloride density gradient is used more often, along with mild conditions for organelle lysis in order to avoid fragmentation of the DNA by mechanical factors.
Continued: Size and conformation of DNA.
