Leaf disks from 24 different species were digested by 5 μl of the enzymatic cocktail in 50 μl of digestion buffer. Thirty μl of liquid containing cell debris were drawn up and released DNA was isolated using Dynabeads® DNA DIRECT™ Universal kit (Dynal). Fig. shows an agarose gel of 25% of the DNA isolated (10 μl). Most DNA are high-yield and of high-molecular weight. The amount of lambda DNA/Hind III loaded into the gel was 250 ng (or 500 ng, bottom half, right). The 23 kb band thus represented approximately 120 ng of DNA. The amount of plant genomic DNA obtained was variable from species to species. For some of them (Humulus lupulus, Vitis vinifera, Narcissus pseudonarcissus, Tilia sp., Lilium henryi and Helleborus dumetorum) the amount of loaded DNA was equal to or higher than 120 ng. As only 25% of the isolated DNA were loaded into the agarose gel, it can be estimated that the method permits the isolation of approximately 50 to 500 ng of genomic DNA from a leaf disk, depending on the species.
Figure 1 Electrophoretic aspect of enzymatically isolated DNA. A: Agarose gel electrophoresis of typical enzymatically isolated DNA from 24 different species (in the following order: Phlomis fructicosa, Humulus lupulus, Veratrum album, Scilla bifolia, Astragalus (more ...)
In the experiment described above, species on which the method was previously tested were selected. In order to empirically examine to what extent the method works on different species, simultaneous extraction of 48 randomly chosen species was carried out in a microtitration plate (as shown on Fig. ) using the Wizard® Magnetic 96 Plant System kit (Promega) to isolate the released DNA (Fig. ). One fourth of the isolated DNA was loaded into the gel. In approximately 75% of the species, genomic DNA was visible. Several DNA extracts show partial degradation. No DNA is visible for Gladiolus palustris, Viburnum farreri, Weigela sp, Prunus padus, Ribes petraeum, Betula sp, Sorbaria sorbifolia, Pelargonium sp, Saintpaulia magungensis.
Enzymatic disruption of leaf disks in a microtitration plate. A flat bottom microtitration plate filled with 50 μl of digestion buffer and leaf disks of different species before the adding of the enzymatic cocktail.
In experiments described above, the cell wall digestion was done overnight for convenience. In order to follow the release of DNA at different times of enzymatic digestion, three 5 mm leaf disks from dry leaves of Ilex aquifolium were digested for 0.5 to 5 hours and the released DNA was isolated with the Wizard® Magnetic 96 Plant System kit (Promega). Fig. shows that some DNA is already released at 0.5 h and that 3 to 4 hours are sufficient to release most of the DNA from this species. A short digestion time (1 to 3 hours) is sufficient for soft leaves such as Arabidopsis, Begonia, Brassica, Beta, Alium, Nicotiana, Triticum, Piper ...etc. However, incubation times need to be increased for Fragaria, Ribes, Oryza, Soya, Zea ...etc. Thus, although 1 to 3 hours are generally enough, the incubation time for a given species is not foreseeable and the appropriate length of digestion has to be determined experimentally before undertaking large-scale DNA isolations.
Time scale DNA release from digesting leaf disks. Triplicate essay of time scale DNA release from leaf disks of Ilex aquifolium at 0.5 to 5 hours of enzymatic digestion. Size marker: lambda/HindIII DNA.
The method is highly reproducible. Fig. shows 16 DNA isolated from dry leaf disks of Aster amellus and Ilex aquifolium and from seeds of Allium porum (cut into 2 pieces, see Materials and Methods), using the Wizard® Magnetic 96 Plant System kit (Promega). Fig. shows a comparison of the amount of DNA isolated from Ilex aquifolium by a CTAB-based extraction method (lines 1–5, the protocol includes a treatment with RNase) and by the enzymatic method described here (lines 6–10) using the Wizard® Magnetic 96 Plant System. In both cases the amount of loaded DNA is one fifth of the DNA corresponding to a leaf disk of approximately 3 mg (dry weight). The amount of isolated DNA is similar for both methods.
Figure 4 Reproducibility of enzymatical isolation of DNA. (A) From leaf disks of 16 individuals of Aster amellus (one tenth of the isolated DNA was loaded), (B) From 16 leaf disks of Ilex aquifolium (one fifth of the isolated DNA was loaded) and (C) From 16 seeds (more ...)
Figure 5 Features and properties of enzymatically isolated DNA. A: comparison of the amount of DNA isolated from Ilex aquifolium leaves by a conventional extraction method (lines 1–5) and by the enzymatic method described here (lines 6–10). In (more ...)
As indicated by Fig. , lambda DNA/Hind III does not show degradation during incubation with the enzymatic mix, nor in the presence of an overnight digesting leaf disk of Ilex at 50°C. This indicates that the digestion mix does not contain active DNase in the condition used for digestion, and that in the case of Ilex endogenous plant DNase are inactivated by the digestion mix.
Fig. also shows PCR amplification of a plastid sequence (Fig. ), a multi-copy nuclear sequence (Fig. ) and a single-copy nuclear sequence (Fig. ) from 1 μl of DNA isolated by the enzymatic method from Ilex aquifolium, Aster amellus and Solanum tuberosum. The primers have been designed for the genus Ilex. Thus the few negative PCR in other species probably result from primer mismatch and not from polymerase inhibition. RAPD amplifications are also shown (Fig. ).
The enzymatic cocktail is produced from Trichoderma longibrachiatum fermentation and could be contaminated with its DNA. Moreover, in the case of a long overnight enzymatic digestion, there is a risk of contamination from bacteria or fungi covering the surface of plant tissues. To examine if such contamination could be a problem, fungi and bacteria specific PCR markers were tested on DNA extracted from (1) a digestion mix alone, or (2) a digestion mix "contaminated" with a Ilex aquifolium leaf disk removed after 10 min and further incubated overnight at 50°C, or (3) a mix digesting a Ilex aquifolium leaf disk overnight at 50°C (Fig. ). No fungus (particularly Trichoderma longibrachiatum) or bacterial template could be detected. Instead, Ilex aquifolium templates are detected. Indeed, the ITS PCR product found in the digestion mix "contaminated" with a Ilex aquifolium leaf disk removed after 10 min and further incubated overnight at 50°C (line 10) has the same size as ITS of Ilex (larger than ITS of Trichoderma). Further sequencing demonstrated that this PCR product was an ITS sequence of Ilex and not of Trichoderma (data not shown). Similarly, the prokaryotic 16S rDNA sequence obtained from the mix digesting an Ilex aquifolium leaf disk overnight at 50°C (line 19) represented the plastid (prokaryotic) 16S rDNA of Ilex (data not shown), and not a bacterial sequence.
Figure 6 Contamination checking: PCR markers for fungi and bacteria in DNA isolated by the enzymatic method. Lines 1 to 12: Internal transcribed spacer (ITS) of ribosomal DNA amplified with eukaryotic specific universal primers ITS1 and ITS4 . Lines 13 to (more ...)