A Simple Protocol for Isolating DNA from Fresh Cucurbita Leaves

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Cucurbit Genetics Cooperative Report 21:46-47 (article 16) 1998

Rebecca Nelson Brown and James E. Myers
Department of Horticulture, Oregon State University, Corvallis, OR 97331

Mark Hutton and Paul Miller
Sunseeds, 8850 59th Ave. NE, Brooks, OR 97305-0982

Introduction. Many DNA isolation protocols have been published, and there are many kits available from scientific suppliers. However, no protocols have been published specifically for use with the Cucurbita species. Most of the available kits are based on the alkaline lysis method of DNA extraction. This method has been found to be unsatisfactory for leaf tissue from all but the youngest squash plants because of the presence of secondary compounds. Many CTAB-based protocols have been published, however most require the leaf tissue to be frozen in liquid nitrogen or lyophilized, resources that are not always available. While polysaccharides have been the most commonly reported problem in DNA extraction from cucumber and melon (1,3), other compounds that bind to the DNA during extraction nd interfere with PCR seem to be more of a problem in squash. The PVP-40 in the extraction buffer effectively binds these compounds (4).

The following protocol was adapted from published CTAB protocols (2,5) with the coals of being able to use fresh tissue and having a relatively fast and simple protocol. The protocol is given for a sample size of approximately 0.5 g of fresh leaf tissue but it can be readily scaled up or down. This is a convenient sample size because it is readily obtained from a seedling 10-14 days after planting without killing the plant, and it yields sufficient DNA for many PCR reactions.

Materials and Methods. For extraction of squash genomic DNA one can use partially expanded leaves from plants of any age. The leaves are ground fresh, without freezing or drying. The best results are obtained if DNA is extracted immediately after the leaves are harvested, but they can be stored in plastic bags in the refrigerator for up to two weeks. the leaves can be ground in grinding buffer in sample grinding pouches (Agdia). The pouches are convenient in that they are sterile, can also be used for collecting and storing samples, and avoid the need to filter the homogenate.

Solutions

  1. Grinding buffer: 0.5 M Tris (pH 8.0), 20 mM EDTA (pH 8.0), 2.0 M NaCl.
  2. DNA extraction buffer: 100 mM Tris (pH 8.0), 20 mM EDTA (pH 8.0), 2 M NaCl, 2% (w/v) CTAB (acetyltrimethylammonium bromide),. (Note: It is important to add the extraction buffer ingredients in the order shown, and to fully dissolve the CTAB before adding the PVP-40, or the mixture will clump. Dissolve the CTAB and the PVP-40 by stirring gently on a hotplate. Store the extraction buffer above 15 C to avoid precipitating the CTAB. Most protocols add α -mercaptoethanol to the extraction buffer, however it appears to be unnecessary for squash.)
  3. Washing buffer: 80% ethanol, 10mM ammonium acetate.
  4. Other reagents: chloroform:isoamyl alcohol (24:1), isopropanol, TE buffer, RNAse A, 95% ethanol, 3M sodium acetate.

Protocol

  1. Preheat the extraction buffer to 65 C. Chill the grinding buffer to 0 C. Grind approximately 0.5g fresh leaf tissue in 2 ml grinding buffer. Transfer to a 10 ml centrifuge tube. Hold samples on ice until adding 2 ml extraction buffer. Shake the tubes gently to mix the buffers and place in a 65 C water bath for 30 minutes with periodic shaking. (Note: it is essential that the extract (sap + buffers) remain at pH 8.0 to prevent precipitation of the DNA).\
  2. Add 3.5 ml chloroform:isoamyl alcohol at room temperature and mix for 10minutes by gentle inversions.
  3. Centrifuge for 10 minutes at 11,000 x g to separate the phases.
  4. Using a sterile plastic transfer pipette, transfer the aqueous layer to a fresh tube containing 2.5 ml ice-cold isopropanol.
  5. A layer of DNA may be immediately visible at the top of the sample. The DNA can be spooled out, but yields will increase if the tubes are inverted gently to mix the sample, and the DNA allowed to precipitate overnight at room temperature.
  6. Centrifuge for 5 minutes at 650 x g to pellet the DNA. The pellet should be off-white or brown.
  7. Pour off the supernatant, drain the tubes on a tissue, and add 8 ml washing buffer. Invert gently to wash the pellet until it is white.
  8. Centrifuge for 10 minutes at 650 x g. Carefully pour off the supernatant and drain the tubes. Air-dry the pellet until no smell of ethanol remains.
  9. Resuspend the pellet in 500 1 of TE buffer. Transfer to a 1.7 ml centrifuge tube. The sample will contain a mixture of DNA and RNA.
  10. If the presence of RNA in the sample will interfere with quantification or use of the DNA, follow steps 11-14:
  11. Add 3 μl RNAse A to each sample. Mix well, and incubate for 15 minutes at 37 C with occasional shaking.
  12. Add 50 μl 3M sodium acetate and 1 ml 95% ethanol to each tube and mix thoroughly. The DNA should be clearly visible.
  13. Centrifuge for 1 minute at maximum speed in a microcentrifuge to pellet the DNA. Pour off the supernatant, drain the tubes and air-dry the pellet.
  14. Resuspend the DNA in 500 μl of TE buffer. Store at 4 C (short term) or at 80 C (long term).

The protocol has been used with leaf tissue from Cucurbita pepo and interspecific C. pepo x C. Moschata hybrids. It should be effective with any Cucurbita species, although the addition of 0.25 mg activated charcoal per gram of tissue may be necessary with species containing higher levels of secondary compounds. Average yield was 240 μg of DNA per gram of leaf tissue with a ratio A/260/A280 between 1.8 and 2.0. These yields are lower than those reported by Baudracco-Amas for melon (1),but much of that can be attributed to the use of fresh rather than dried tissue. The DNA is not degraded, is in pieces of approximately 4000 bp each, digests to completion and amplifies in the PCR process.

Literature Cited

  1. Baudracco-Arnas, S. 1995. A simple and inexpensive method for DNA extraction from Cucumis melo L. Cucurbit Genet. Coop.Rept. 18:50-51.
  2. Bi, I. Vroh, et al. 1996. Improved RAPD amplificaiton of recalcitrant plant DNA by the use of activated charcoal during DNA extraction. Plant Breeding 115:205-206.
  3. Fang, G., S. Hammar and G. Grumet. 1992. A quick and inexpensive method for removing polysaccharides from plant genomic DNA. Biotechniques 13:52-56.
  4. Lodhi, M.A., et al. 1994. A simple and effective method for DNA extraction from grapevine cultivars and Vitis species. Plant Mol. Bio. Rept. 12:6-13.
  5. Steenkamp, J., et al. 1994. Improved method for DNA extraction from Vitis vinefera. Am. J. Enol. Vitic. 45:102-106.