Cucurbit Genetics Cooperative Report 18:50-51 (article 24) 1995
A Simple and Inexpensive Method for DNA Extraction from Cucumis melo L.
INRA, Station d' Amelioration des Plantes Maraicheres, BP 94, 84143 Montfavet Cedex, France
The applications of current nucleic acid technologies to crop improvement include gene mapping, genetic fingerprinting, population studies and phylogenetic analyses. These techniques have application for the improvement of melon (Cucumis melo L.). This species is one of the most important vegetable crops in the world, but few molecular biology studies have been published. Phylogenetic studies have been recently performed using RFLP markers (7).
DNA extraction for breeding purpose needs to be simple, rapid and inexpensive. We tried various methods for extracting DNA from melon (1, 3, 8) including modifications of these methods. However, DNA extraction was unusable because sticky contaminants, probably polysaccharides, were not removed. The method of Liechtenstein and Drapper (4) was modified and good quality DNA was obtained from cotyledons and leaves of plants grown in a greenhouse. This DNA is suitable for restriction digestion, hybridization and amplification in the polymerase chain reaction.
Materials and Methods: for extraction of melon genomic DNA, we used young leaves or cotyledons harvested and dried in a food dehydrator at 30C for 24 to 36 hours (8) and stored at -20C until use.
Extraction buffer :
10 mM sodium EDTA, 50 mM Tris-HCl pH-8.0 0.7 M NaCl. 1% CTAB (acetyltrimethylammonium bromide), 1% (w/v) β -mercaptoethanol (
β -ME). The solution was made up without
β -ME on a heated stirrer avoiding foaming. It should be autoclaved. The
β -ME is added just before use.
Chloroform:octonol: Chloroform :octonol 24:1 (v/v)
CTAB:NaCl : CTAB 10% (w/v), 0.7 M NaCl
Precipitation buffer: 10 mM sodium EDTA, 50 mM Tris-HCl pH_8.0. 1% CTAB, RNAase A 10 mg/,L.
Ethanol:Acetate: Ethanol 76% (v/v), sodium acetate 0.2 M
TE buffer: 1 mM sodium EDTA, 10 mM Tris-HCl pH-8.0 and autoclaved.
- Grind 1.0 g of dried leaves or cotyledons in a fine herb electric mill (Moulinex 534) to a very find powder. This is probably the most important step in efficient disruption of the plant cell wall and the key for good DNA recovery. It is possible to store this powder at -20C until use.
- Tip the powder into a 50 mL polypropylene centrifuge tube. Add 15.0 mL of extraction buffer (at 56C), cap the tube and mix gently by inversion.
- Incubate in water bath at 56C for 20 min, occasionally agitating the tube gently to keep the extract mixed.
- Allow the incubation mixture to cool to room temperature. The temperature should not fall below 16C as precipitation of CTAB will occur.
- Add 15.0 mL of chloroform:octanol. Cap the tube and mix by gently inverting the tube 20 to 25 times to form an emulsion.
- Pellet the debris and separate out the organic and aqueous phase by centrifugation at 3,000xg for 20 min at 20C.
- Pour off the aqueous phase (top layer) into a clean 50 ml. centrifuge tube.
- Add 2.0 mL of CTAV:NaCl and mix gently. Add 15.0 ml. of chloroform:octanol, mix by gentle inversion until one phase emulsion (white or yellow colour) forms and separate the new aqueous phase by centrifugation at 3,000xg for 20 min at 20C.
- Pour off the supernatant into a new clean 50 mL centrifuge tube containing 15.0 mL of precipitation buffer, avoiding the interphasic debris. Mix gently and leave to stand at room temperature for one hour while the precipitate forms.
- Pellet the precipitate at 1.500xg for10 min at room temperature. Do not pellet the precipitate too hard as a compact pellet is difficult to redissolve. In a good preparation the pellet should be whitish or slightly discoloured, but sometimes at this step, the pellet may be yellowish.. This colour will disappear with the RNAase A step.
- Drain the pellet by inverting the tube, held in a rack, onto a paper towel, for 2 min.
- Dissolve the nucleic acid in CTAB pellet with 2 mL NaCl 1.0 M. If the pellet is too hard to dissolve, heat to 56C for a few minutes until dissolution.
- When the pellet is fully dissolved, add 30 L of RNAase A and incubate at 37C for half to one hour.
- Add two volumes of freezed (-20C) absolute ethanol, mix by gentle inversion until DNA strands begin to appear.
- With a 'Pasteur hook' take the DNA strands and wash 2.0 mL ethanol:acetate for 10 min. At this step, the DNA extract should be white.
- Drain the DNA strands and put into a sterile microfuge tube with 200 - 400 L of TE buffer.
- Quantify DNA in a spectrophotometer at A260 or in an agarose gel with a phage scale of concentration.
- DNA can be stored at -20C over months and at -80C over years.
Results and discussion: DNA yield from C. melo by this procedure ranges from 0.25 mg/g of dried leaf or cotyledons tissues with a ratio A260/A280 between 1.8 and 2.0. The procedure is simple and fast, and 36 to 48 DNA samples may be processed in a single day. Sufficient quantities of DNA were obtained from 10 grams of fresh leaf for large scale RFLP or RAPD analyses. The native DNA was not degraded, and the digestion by restriction endonuclease was complete. This CTAB-based procedure used for DNA extraction is modified from Liechtenstein and Draper (4), and does not involve centrifugation in a CsCl gradient. This technique does not use liquid nitrogen to assist in the grinding of plant material, the plant tissues being dehydrated and ground in a fine herb mill. this method is easier and less expensive than the original one. It is possible to store ground dehydrated tissue for a long time at -20 C until use. Sample of DNA extracted from one gram of dried tissue cost approximately $1 U.S. with this method.
The polysaccharides are difficult to separate from DNA (6). These compounds are easily identifiable in the DNA preparation as they result in a sticky, viscous consistency to the DNA preparation, making it difficult to dissolve in TE buffer. Polysaccharides interfere with several enzymes such as polymerases, ligases and restriction endonucleases (5). Fang et al. (2) found that 1 M NaCl facilitated the removal of polysaccharides by increasing their solubility in ethanol. In our method, three CTAB steps facilitated the removal of polysaccharides, and the final addition of 1 M NaCl facilitated the DNA solubility in TE buffer. Complete digestion with restriction endonucleases and amplification in PCR indicate a good elimination of polysaccharides in DNA samples.
In melon, because a few intracellular RNAase exists, a large quantity of RNA was extracted with the DNA. Because of this RNA interferes with spectrophotometer quantification, a digestion with Nasser A proved to be necessary.
We have used DNA prepared by this method in a number of molecular marker-based studies of C. melo, including analysis of genetic diversity and mapping using RFLP and RAPD markers.
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