Chapter III

Rapid DNA Extraction from Plants

1 Introduction The first step of RAPD fingerprinting is the preparation of the target DNA template. Intuitively, minimal DNA template preparation should be necessary for RAPDs since, theoretically, PCR may amplify a single DNA molecule. It seems one would simply homogenize tissue and allow the PCR to "find" and amplify the target DNA. Indeed, many rapid DNA isolation methods designed for use with PCR actually involve little isolation of DNA. Rather, they employ a "grind and use" process (e.g.,Wang et al. 1993) or minimal purification (e.g., Edwards et al. 1991).These methods are very fast, require little tissue and amplify well with plants that are amenable to DNA extraction and that contain few interfering secondary metabolites. However, the resulting DNA templates are not very pure and may not be stable for long periods of time. Recently, DNA purity has been implicated as one of the most important factors in RAPD reproducibility (McClelland and Welsh 1994). McClelland and Welsh (1994) suggest that researchers use only high quality templates to assure reproducible RAPDs. The assay they suggest consists of replicate RAPD PCRs in which the DNA concentration is titrated over two orders of magnitude. If the DNA is of adequate quality the replicates should yield identical RAPD fingerprints. In addition, Heinze (1994) found that the addition of RNAse A improved RAPD reproducibility with gymnosperm embryos. However, the basal method was a crude prep similar to that of Edwards et al. (1991). Thus, it seems-likely that there could be several interactive factors involving DNA purity or lack thereof in RAPD reproducibility. It seemsprudent to researchersperforming RAPD PCR to assure themselves that the isolated DNA is of sufficient quality to ensure reproducible RAPD fingerprints. The objective of this chapter is to describe a rapid CTAB miniprep that yields relatively pure DNA from plants and that can also be used

with fungi and fish. The procedure features the cationic detergent CTAB (hexadecyltrimethylammonium bromide) in the homogenization buffer, one chloroform extraction, and an alcohol precipitation. This procedure was published by Stewart and Via (1993) and has its origins in previously published procedures (Murray and Thompson 1980; Doyle and Doyle 1987, 1990). The procedure described in this chapter further streamlines the Stewart and Via {1993) miniprep procedure. 2 Materials Equipment Supplies cordless electric drill capable of speeds between sob and 1000rpm (e.g., Black and Decker CD 2000 "Ranger") disposable pellet pestles (e.g., Bio- Ventures catalog# P-50 Murfreesboro, Tennessee,USA). Alternatively, one may use ( 1000~l plastic pipette tip that has been pushed onto a deburrinj tool as a homogenizing pestle (Stewart and Via 1993).The end 0 the pipette tip is crimped upward when pressed on the tool (th tip is pressed against the bottom of the pipette tip box), thereb creating a "blade" for homogenization. homogenization buffer (amounts stated are for 11; all reagen1 may be purchased through Sigma Chemicals):100mM Tris-H( (15.76gin 800ml of water, pH adjusted to 8.0 with NaOH), 20 ml EDTA (40 ml of 0.5 M EDTA, pH 8.0), 2 % w/v CTAB (20 g), 1:42 J NaCl (81.8g), 2 % w/v PVP-40 (polyvinylpyrrolidone, averaj molecular weight 40000) (20 g), 5 mM ascorbic acid (0.88 ~ 4.0 mM DIECA (diethyldithiocarbamic acid) (0.69g). Bring vc ume to 11. This solution may be kept indefinitely at room tempe ature. Just prior to use, add 6 ~l 2-mercaptoethanol per ml homogenization buffer. extraction solution: chloroform:isoamyl alcohol (24:1). This m be stored indefinitely at room temperature in a sealed containe ethanol or isopropanol 3 Experimental Procedure 1. Harvest between 0.02 and 0.2 g fresh leaf, callus, embryo, sti (or appropriate animal tissue) into a microfuge tube. Place on j These may be sufficiently stable for hours to days, depend upon species.

Reagents and solutions

Note: For plants, best quality and yield is obtained when newly flushing leaves are used. However, the procedure has been used with a plethora of tissues, including herbarium specimens. Caution: All subsequent steps should be performed in a fume hood. 2. Aliquot 500 ~l of homogenization buffer (including the 2mercaptoethanol) into each microfuge tube. For tough leaves from woody plants (exemplified by oaks or cranberry) heating the buffer to 65C may increase yield. 3. Chuck pestle directly onto drill and homogenize tissue. The disposable pestles may be r~used after decontamination with 100/<1 commercial bleach followed by subsequentwashes with water. 4. Incubate tubes at 65C for 5-60 min. The incubation time is not crucial for most species and can be determined by investigator convenience. 5. Add 500 ~l extraction solution to tubes and mix. For most plant speciesthis may consist of ten inversions. For recalcitrant species (such as woody plants) placing tubes horizontally on a shaker (500 rpm) for 5-10 min may increase yield. 6. Centrifuge at 3000g for 5 min. Slightly higher g or longer centrifugation periods may be necessaryto keep the interface compact and the top layer clear if many (>12) samples are centrifuged at a time. 7. Pipette top (aqueous) layer (about 400 ~l) into a fresh tube. 8. Add 800 ~l ethanol or 270 ~l isopropanol and invert several times to precipitate DNA. The sample may be allowed to set at room temperature or 4C for 5-60 min to increase DNA yield. Longer periods will not affect DNA quality, but may slightly increaseyield. 9. Centrifuge at 13000-16000 g for 10-20 min at room temperature. 10. Decant supernatant, air-dry pellet, and resuspend in 50-500 ~l water. 4 Resultsand Comments This method has been used with successin several plants (Arabidopsis, banana, rapid cycling Brassicas,canola, chestnut, cranberry, and tobacco, to name a few), fungi (such as Russula spp.) and fish (such

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C. NEALSTEWART, III Rapid DNA Extraction JR.:

from Plants

as Rivulus marmoratus). DNA yields, as quantified using Hoechst 33258 dye fluorometry, are typically between land 50!!g depending on species, starting material and extraction conditions mentioned earlier. This represents enough DNA for between 40 and 2000 RAPD PCRs. RAPD fingerprints using this method compare favorably to those using very pure DNA isolated by the method of Doyle and Doyle (1990) and subsequent purification through a CsCI ethidium bromide gradient (Stewart and Via 1993). This method has recently been used to isolate DNA for restriction digests and subsequent Southern blotting (e.g., our lab; May et al. 1995). Restrictability is another indication of high quality DNA. The prepared DNA is stable in time. For example, cranberry DNA templates that were extracted over 2 years ago and stored at -20C yield identical RAPD fingerprints now as when the DNA was first extracted. In addition, they continue to pass the McClelland and Welsh (1994) test and produce reproducible RAPD fingerprints when template amounts are varied over two orders of magnitude (5-500 ng). In summary, the CTAB miniprep method is rapid (one person can process 100-200 samples in a typical workday) and yields adequately pure DNA for RAPD fingerprinting. References
Doyle JJ,Doyle JL (1987) A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochem Bull 19:11-15 Doyle JJ,Doyle JL (1990) Isolation of plant DNA from fresh tissue. Focus 12:13-15 Edwards K, Johnstone C, Thompson C (1991) A simple and rapid method for the preparation of genomic plant DNA for PCR analysis. Nucleic Acids Res 19:1349 Heinze B (1994)RAPD reactions from crude plant DNA. Mol Biotechnoll:307-310 May GD, Afza R, Mason HS, Wiecko A, Novak FJ, Arntzen CJ (1995) Generation of transgenic banana (Musa acuminata) plants via Agrobacterium-mediated transformation. Bio/Technology 13:486-492 McClelland M, Welsh J (1994) DNA fingerprinting by arbitrarily primed PCR. PCR Methods Applic 4:s59-s65 Murray HG, Thompson WF (1980) Rapid isolation of high molecular weight DNA. Nucleic Acids Res 8:4321-4325 '.. Stewart CN Jr, Via LE (1993)A rapid CTAB DNA isolation technique useful for RAPD fingerprinting and other PCR applications. BioTechniques 14:748-751 Wang H, Qi M, Cutler AJ (1993) A simple method of preparing plant samples for PCR. Nucleic Acids Res 21:4153-4154 .