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J Appl Genet 50(2), 2009, pp.

105–107

Short communication

High-quality plant DNA extraction for PCR: an easy approach
I. Ahmed1,2, M. Islam1,3, W. Arshad1, A. Mannan1, W. Ahmad1, B. Mirza1
1 2

Department of Biochemistry, Quaid-i-Azam University Islamabad, Pakistan Allan Wilson Centre for Molecular Ecology and Evolution, Massey University, Palmerston North, New Zealand 3 Department of Genetics, Hazara University, Mansehra, Pakistan

Abstract. Polymerase chain reaction has found wide applications in modern research involving transformations and other genomic studies. For reproducible PCR results, however, the quantity and quality of template DNA is of considerable importance. A simple and efficient plant DNA extraction procedure for isolation of high-quality DNA from plant tissues is presented here. It requires maceration of plant tissue of about 1.0 cm2 (e.g. of a leaf blade) in DNA extraction buffer (100 mM Tris-HCl, 100 mM EDTA, 250 mM NaCl) using 1.5-mL microfuge tubes, followed by cell lysis with 20% SDS, and DNA extraction with phenol: chloroform: iso-amyl alcohol (25:24:1). Hydrated ether is then used to remove polysaccharides and other contaminants from the DNA preparation. Average DNA yield is 20–30 µg cm–2 for fresh tissues, and ratio of absorbance at 260 nm to absorbance at 280 nm is 1.5–1.8. The DNA is quite suitable for PCR using microsatellites, RAPD and specific markers for recombinant selection. Amplifications have been obtained for these markers by using template DNA extracted from fresh as well as frozen leaf tissues of various plants, including barley, oat, potato and tomato. DNA stored for more than 2 years has been successfully amplified with microsatellite markers, which shows suitability of this method after long-term storage of DNA. Besides, the ease of use and cost-effectiveness make the procedure attractive.
Keywords: DNA extraction, hydrated ether, microsatellites, phenol, chloroform, isoamyl alcohol, plant tissues, polysaccharides.

Polymerase chain reaction (PCR) has revolutionized the genetic and molecular biology studies since its introduction. However, successful amplification with reproducible results in PCR, depends upon the quality and quantity of template DNA. Many plant DNA extraction protocols are known, including CTAB and salt extraction method (Doyle and Doyle 1987) and its modification (Huang et al. 2000). Conventional DNA extraction protocols (Jobes et al. 1995; Cheng et al. 2003) require large quantities of tissue (in grams) to be ground (with a pestle and mortar), which requires long time for plant growth, and thus are not time-efficient if the tissue is to be recovered after seed germination. Some methods (see Sharma et al. 2002) use liquid nitrogen, even though the

use of liquid nitrogen is considered not to be safe. Other protocols require small quantities of tissues, but also have associated limitations, such as the use of specialized apparatus, like a matrix mill (Hill-Ambroz et al. 2002), 96-well microtitre plates and centrifuges with a rotor for microtitre plates (Mogg and Bond 2003), or use of commercially available DNA extraction kits, which have a high cost per sample ratio. Moreover, many DNA extraction protocols result with high polysaccharide contaminations, the potential inhibitors of enzyme reactions (Fang et al. 1992; Porebski et al. 1997; Schlink and Reski 2002). We are interested in Agrobacterium and biolistic gene-gun-mediated transformation of tomato, potato and rice. In addition, we are also in-

Received: September 11, 2008. Accepted: December 16, 2008. Correspondence: I. Ahmed, Allan Wilson Centre for Molecular Ecology and Evolution, Massey University, Palmerston North, New Zealand; e-mail: I.Ahmed@massey.ac.nz; iaqureshi_qau@yahoo.com

and also suitable for long-term storage of DNA samples. which is expected to damage the DNA.8. 6. Repeat steps 9 and 10 to eliminate remaining impurities from the DNA preparation. Samples 1–6 shown in the figure are treated with hydrated ether. Dissolve these impurities in ether by vigorous shaking of the microfuge tubes. With our protocol. 10. prepare appropriate dilutions for downstream applications. we focused on optimizing a rapid plant DNA extraction protocol without the above-mentioned limitations.8% agarose gel containing DNA extracted from barley leaf tissues is shown in the figure 1. while samples 7–12 are without ether treatment. To meet our requirements. Add 250 µL of phenol : chloroform : isoamyl alcohol (25:24:1) to the sample. 1 mM EDTA) or distilled water in equal proportions].000 rpm for 1 min. this method avoids DNA precipitations with ethanol. terested in an analysis of genetic diversity in plant species. Ether. 1994. which reflects the importance of the purification step with ether. 9. As shown in the gel. Carefully remove and discard the upper organic phase (ether). with plant tissue debris deposited at the interphase of the two phases. thus avoiding possible downstream inhibitions of PCR with ethanol re- . However. We used this protocol for DNA extraction from transgenic tomato. hydrated ether efficiently removes the polysaccharides present in the samples.8% agarose gel. which could be used for a large number of fresh and frozen plant samples belonging to different species. Sharma et al. 8. Here we report the protocol in a convenient way.g. 3. 2002) because of freezing and thawing. 11. Similarly. potato and rice. One of the major problems commonly associated with DNA is that it does not give reproducible PCR results after long-term storage (Lodhi et al. Add 50 µL of DNA extraction buffer to the tube and macerate the tissue with a hand-operated homogenizer. Cut a small piece of plant tissue. The impurities removed by ether have a significant absorbance in the UV region (260 nm. on the other hand. 5. This procedure has been used in our laboratory for the last 3 years. Remove traces of ether by air-drying at room temperature for 30 min or at 35°C for 10 min. a leaf (~ 1 cm2) and put it into a 1. A white-coloured interphase. Collect the upper aqueous phase into a new microfuge tube (take care to prevent its mixing with tissue debris in the interphase). The ratio of absorbance at 260 nm to absorbance at 280 nm (R260/280) varies between 1. Ahmed et al. showing reliability and suitability of this method for long-term storage of DNA. The interphase is dissolved and consequently removed with ether. Mix aqueous and organic phases manually. might have some inhibitory effects on DNA digestion with restriction enzymes. Primers specific to rol C. NPT-II. Impurities will collect at the interface of the two phases immediately after ether addition.106 I. Turn on a water bath or heating block and set temperature at 65°C. Centrifuge at 8. Aqueous and organic phases will become clearly separate. and Artemisia sp. wild oat. which is an indicator of the purity of DNA. HAL-I and HAL-II genes were used for successful amplification. even more than 2-year-old DNA dilutions have been used successfully for microsatellite amplifications. This protocol proposes significant modifications of the method suggested by Kang and Yang (2004). using SSR and RAPD markers. Visualize DNA on 0. 2. 21 microsatellite markers in oat and barley have been amplified using template DNA extracted with this protocol. and then discard the interphase and organic phase. A representative 0.000 rpm for 7 min.5 and 1. however. by vigorous shaking and inverting the microfuge tubes for 10 s (because vortexing may cause mechanical shearing or physical damage to DNA). efficient in terms of time and cost. appears between aqueous and organic phases just after addition of ether. 12. 7. Add 250 µL of hydrated ether to the DNA preparation [hydrated ether is prepared by mixing ether with Tris EDTA (10 mM Tris. Bring the volume of DNA extraction buffer to 250 µL and thoroughly homogenize the tissue in buffer. 280 nm) when present in the samples. representing polysaccharides and possibly phenolic residues.5-mL microfuge tube. Store DNA at –20°C for future use. Besides other advantages. Add 25 µL of 20% SDS and vortex for 30 s. as we could not obtain restriction digestion.. A comparison of the average amount and purity of DNA for the same samples with and without ether purification is given in Table 1. Centrifuge at 14. 4. and thus lead to erroneous results in DNA quantifications. e. so that it is easy to follow: 1. there is no visible difference in appearance of the samples with and without ether treatment. Quantify DNA by using UV-spectrophotometer. including barley. Put the microfuge tube at 65oC for 20–60 min for cell lysis (longer incubation gives a higher yield of DNA).

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