Advances in Biological Research 2 (5-6): 83-89, 2008 ISSN 1992-0067 IDOSI Publications, 2008

Protocol for Isolation of Genomic Dna from Dry and Fresh Leaves of Vigna Species Suitable for Rapd and Restriction Digestion
K. Choudhary, N. Mathur, O.P. Choudhary and U. Pillai Department of Biotechnology, Lachoo Memorial College of Science and Technology, Jodhpur, India
Abstract: A simple and efficient protocol for isolating genomic DNA from fresh and dry leaves of Vigna aconitifolia and V. trilobata was developed. It involves a modified CTAB procedure using 3% CTAB, 4% -mercaptoethanol, 2 M NaCl and 5% PVP. The extraction was carried out at 70C. A slight increase in the concentrations of these chemical components and temperature helped in the removal of secondary metabolites and polysaccharides from the DNA preparation. The quantity and purity of isolated DNA was higher when compared with DNA extracted by the methods of Dellaporta et al. (1983) and Doyle and Doyle (1990) [9, 13]. The DNA yield ranged from 45 -55 g per g of leave samples and it was 1.25 times greater in dried than fresh samples. The DNA samples were found suitable for analysis with restriction enzyme digestion and randomamplification of polymorphic DNA (RAPD). Key words: DNA isolation Leaves Legume PCR amplification Polyamines PVP Secondary metabolite INTRODUCTION The application of DNA technology in agricultural research has progressed rapidly over the last twenty years, especially in the area of cultivar identification and characterization as well as determination of population diversity in many plant species [1-5]. The application of this powerful tool in some plant species has however been constrained by lack of efficient nucleic acids isolation techniques. The extraction of the nucleic acids is difficult in a variety of plants because of the presence of polyphenols and secondary metabolites that interfere with DNA isolation procedures and reactions such as DNA restriction, amplification and cloning [6]. A large number of secondary metabolites such as tannins, alkaloids, phenolics and terpens responsible for the valuable pharmacokinetic properties of medicinal plants which interfere with the isolation process, tend to copurify with DNA and interact irreversibly with proteins and nucleic acids [7]. Problems encountered in the isolation and purification of high molecular weight DNA from certain legume plant species include: degradation of DNA due to endonucleases, co-isolation of highly viscous polysaccharides and inhibitor compounds like polyphenols and other secondary metabolites which directly or indirectly interfere with subsequent enzymatic reactions [8]. A good extraction procedure for the isolation of DNA should yield adequate and intact DNA of reasonable purity. Various protocols for DNA extraction have successfully been applied to many plant species [9-13]. The major differences in these protocols mainly concern the ingredients (and also the pH) of the extraction buffer. For example EDTA is generally included in DNA isolation buffers and storage solutions, since this compound chelates bivalent cations and thereby inhibits metal-dependent DNases. Reducing agents such as bmercaptoethanol/dithiothreitol are also usually included in inhibiting oxidation process, which either directly or indirectly cause damage to DNA. Each plant species may require its relevant protocol depending on the demand of the level of DNA purity. For example, Doyle and Doyle (1990) have used CTAB to isolate DNA with the reducing agent -mercaptoethanol in addition to proteinase K which removes protein [9]. Others like Reichardt and Rogers (1993) have used high CTAB concentration which is an active detergent to deter DNase activity and also removes polysaccharides [14]. Edwards et al. (1991) have used SDS and phenol instead of CTAB as a detergent for the same function of pure DNA isolation [11]. These are sometimes further modified to provide DNA suitable for several kinds of analyses [12,15]. The biochemical composition of plant tissues and species varies considerably; therefore it is virtually impossible to supply

Corresponding Author: Dr. Kailash Choudhary, Department of Biotechnology, Lachoo Memorial college for Science and Technology, Jodhpur, Rajasthan-342001, India 83

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a single isolation protocol which is optimally suited for each plant species. Thus, different plant taxa often may not permit optimal DNA yields from one isolation protocol [8]. Even closely related species may require quite different isolation procedures [8]. In addition to a reliable DNA extraction method, the storage of plant tissues for DNA extraction is also important. Most of the protocols recommend isolation of DNA from fresh tissues, but sometimes the samples collected from remote and rare locations may consist of plant parts in dry or semi-dry conditions [16]. This necessitates the development of the protocols for isolation DNA from different plant organs, including dry tissues. Therefore, under these conditions, a good extraction procedure and good plant tissue storage conditions are important in order to extract good quality and quantity of DNA. CTAB is a cationic detergent, which solubilises membranes and forms a complex with DNA [6]. SDS extraction protocol is just but a modified version of CTAB with various alterations to increase the efficiency of removing proteins from the extracted DNA. In the protocol, there is the use of dithiothreitol (DTT), which reduces proteins at millimolar levels requiring only a slight excess of iodoacetamide (or N-ethylmaleimide) to alkylate. SDS is a detergent. It is (or a close relative, of sodium lauryl sulphate) often found in everyday shampoos, where it solubilises grease and oils. In the DNA preparation, it breaks up the lipids in the membranes to free the DNA from the cell. DTT is the common name for a small molecule redox reagent known as Clelands reagent. DTT is an unusually strong reducing agent, owing to its high conformational propensity to form a six-member ring with an internal disulfide bond. DTT is used as a reducing agent for thiolated DNA. The terminal surfur atoms of thiolated DNA have a tendency to form dimmers in solution, especially in the presence of oxygen. DNA isolation protocols generally use CTAB to avoid co-purifying polysacharrides from plant tissues. Keeping this in mind and the fact that Vigna samples carry high amounts of polyphenols and polysacharrides, the standard CTAB method [9]was tried in the experiments. Several modifications including use of dithiothreitol 6.5 mM instead of 2-mercaptoethanol were also evaluated. Other alterations tried included replacing CTAB with SDS [11,17]. Polyvinylpolypyrrolidone (PVPP) has also been used successfully to remove polyphenols along with a high molar concentration of NaCl to inhibit coprecipitation of polysaccharides and DNA. Therefore, SDS method was used also as proposed by Khanuja et al. (1999) [16].
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Plant species belonging to the same or related genera or variety can exhibit enormous variability in the complexity of pathways of dispensable functions. Thus, the biochemical composition of plant tissue of different species or varieties is expected to vary considerably. The chemotypic heterogeneity among species or varieties may not permit optimal DNA yields from one isolation protocol and perhaps even closely related species may require different isolation protocols [8]. Vigna aconitifolia (Jacq.) Marechal, commonly known as the Moth bean, Mat bean, Turkish gram, is native to India and Pakistan belongs to family Fabaceae. It is extensively grown for food in the arid and semi-arid regions of India. It is also an important pulse crop in semi-arid regions adjoining tropical dry regions. Vigna trilobata (L.) Verdc. is a wild species belonging to family Fabaceae. It is commonly called as African gram, three-lobe-leaf cowpea (English); Chidi moth, (Hindi). It is sown in India as a short-term pasture and green manure crop. V. trilobata had a higher content of crude protein than the commonly consumed Indian pulses [18]. Genus Vigna have high amount of polyphenol, orthohydroxyphenols and polysaccharides. These are powerful oxidising agents to interfere with genomic DNA. Some varieties are recalcitrant to inhibit the PCR amplification. DNA isolation protocols for Cicer have been reported by Chakraborti et al. (2006) and from nodules of legumes have been reported by KrasovaWade (2007) [19, 20]. Here, we report an efficient protocol from fresh and dried leaves of V. aconitifolia and V. trilobata for isolation and purification of genomic DNA for RFLP and RAPD analysis. MATERIALS AND METHODS Plant Material: Sample of fresh juvenile leaves were harvested from in vitro grown plantlets raised from seeds of V. aconotifolia and V. trilobata maintained in growth rooms. These were washed with sterilize distilled water followed by 80% alcohol. Samples were divided into two-one was kept at 4C and the other was dried at 50C. The fresh and dried samples were subsequently processed for DNA extraction. Solutions: The extraction buffer consisted of CTAB3%(w/v); Tris HCl (pH 8)- 100mM; NaCl - 2M; EDTA (pH8)-25mM; -Mercaptoethanol-4%(v/v); PVP- 5%(w/v) was prepared.

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The following solutions were also prepared and stored at 4C- Chloroform: Isoamylalcohol-24:1(v/v); TE buffer (10mM Tris HCl, pH8.1Mm EDTA); 80% Ethanol; Ribonuclease A (10mg/ml) DNA Isolation: Fresh and dried leaves of V. aconitifolia and V. trilobata were surface sterilized with sterile distilled water followed by 80% ethanol. The leaves were cut into small pieces of size approx. 1 mm with sterile blade. The pre-chilled mortar and pestle was used to ground fresh (2g) and dried (1g) leaf samples. The powder was then transferred in 8 ml of the extraction buffer (pH 8) into a 30ml centrifuge tube. The mixture was incubated at temperature 70C for 30 min. After incubation the mixture was cooled at room temperature and then equal volume of mixture of chloroform: isoamylalcohol (24:1) was added. Purification of DNA: The samples isolated using the above methods were purified as detailed. To each tube, 500 ml chloroform: iso-amylalcohol (24:1) was added and the contents mixed by shaking for 15 min,followed by centrifugation at 12000 rpm for 15 min. The aqueous phase was transferred to a new tube and then 200 ml 1M NaClTE added to the old tube and shaken for 15 min. The old tube was centrifuged for 15 min at 12000 rpm. The aqueous phase was transferred to the new tube and mixed, followed by centrifugation at 12000 rpm for 15 min in order to settle any remaining debris. The supernatant was then transferred to a new tube. Ice cold isopropanol (700 ml) was added to the sample and mixed gently and centrifuged at 10,000 rpm for 5 min and the supernatant discarded. Cold 75% ethanol and 5M Nacl was added to the pellet to wash it thrice and contents centrifuged at 5000 rpm for 5 min. The ethanol was discarded and the pellet air dried. The pellet was re-suspended in 200 ml 1X TE buffer and incubated overnight at 55C. Rnase Treatment: The DNA was treated with DNase free Ribonuclease A. Large amounts of RNA in the sample can chelate Mg2+ and reduce the yield of the PCR. This step removes RNA from the isolated genomic DNA. RNase (10 l of 10 mg/ml) was added to 100 l of re-suspended DNA pellet and then incubated at 37 C over night. Equal volume of ice-cold absolute ethanol was added to each sample and then centrifuged at 10,000 rpm for 10 min to re-precipitate the DNA. This was done twice. The supernatant was poured off and the DNA pellets air-dried and finally the DNA pellet was dissolved in 50 l of 1xTE buffer.
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Quantification of Genomic DNA: DNA concentration was determined using bio spectrophotometer by measurement of optical density. For this, 10 l of DNA sample was diluted with 100 l of 1X TE buffer (10 X dilution) and the optical density was measured at 260 nm against a 1X TE buffer in a Bio-Spectrophotometer (BL-198, Elico Ltd., India). The DNA concentration was then calculated according to the known method. Optical density (O.D) values were also taken at 280 nm (corresponding to protein), 230 nm (corresponding to RNA) and 320nm (contamination). Total DNA purity was tested by a ratio of O.D values at 230: 260: 280. Quality of the Isolated DNA and Restriction Digestion: The DNA was examined for intactness using the gel electrophoresis method. Two microliter of the isolated DNA, 7 l of sterile distilled water were mixed with 1.0 l of 10 X loading dye and was loaded in a lane of 1% (w/v) agarose gel containing 0.05 l gml 1 of ethidium bromide for checking the quality of the DNA. Hind III digested lambda DNA served as a size marker. The agarose gel electrophoresis was carried out for ~1 hr at 50V. The gel was visualized and photographed under UV light. The purity of extracted DNA was checked with restriction digestion of the purified DNA samples with EcoRI and Hind III (4 unit per g) for 5 h incubation periods and restricted DNA run on ethidium bromide stained agarose gel (1.2%) with EcoR1 digested lambda DNA as marker. PCR Amplifications: To check the quality of isolated DNA from the root samples of selected Vigna species, Polymerase chain reaction (PCR) was performed. PCR were carried out in a final volume of 25 l containing 20 ng template DNA, 100 M each deoxynucleotide triphosphate, 20 ng of random primers: 5 GTCGCCGTCA 3 (P1) and 5 GTTTCGGTCC 3 (P2) (Imperial) in 25 ml volume, 1.5mM MgCl2, 1 x Taq buffer ([10mM] Tris-HCl [pH -9.0], 50mM KCl, 0.01 % gelatin) and 1 U Taq DNA polymerase (M/S Bangalore Genei, India). Amplification was achieved in a thermal cycler (Eppendorf) programmed for a preliminary 4 min denaturation step at 94C, followed by 30 cycles of denaturation at 94C for 1min, annealing at 37C for 1min and extension at 72C for 2 min, finally at 72C for 10 min. Amplification products were separated alongside EcoRI/ Hind III double digested lambda genome as marker (M/S Bangalore Genie, India) by electrophoresis on 1.0 % agarose gels run in 0.5 X TAE (Tris Acetate EDTA) buffer, stained with ethidium

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bromide and visualized under UV light. Gel photographs were scanned through Gel Doc System (Vilber Lourmat, Germany) and the amplification product sizes were evaluated by using the software (Vision Capt Version 14.3) RESULT AND DISCUSSION In case of Vigna species, the isolation of genomic DNA and subsequent PCR amplification were complicated due to abundance of polyphenolics, polysaccharides, RNA and other secondary products. Polyphenols and other plant compounds, cause damage to DNA and/or inhibit restriction enzymes and Taq polymerases. These compounds bind to DNA when cells are lysed [9]. Cell disruption by grinding in liquid nitrogen and incubation in extraction buffer results in lysis of both the cells containing the phenolics and the organelles. In a considerable number of plant species, DNA preparation tends to be brown coloured due to the oxidation of polyphenols to quinonic compounds. These are powerful oxidizing agents that damage DNA and protein [21]. As a consequence, yield of high molecular weight DNA from plants is often very poor. Even in the presence of solublepolyvinylpyrrolidone (e.g. PVP- 40) and polyphenol absorbants such as bovine serum albumin (BSA) in the isolation buffer at concentrations of 1-4 %, the phenolics adhere to DNA in solution, forming a coloured matrix around the DNA. Further, the inclusion of phenoloxidase inhibitors such as diethyldithiocarbonic acid (DIECA), antioxidants (such as sodium bisulfite, cystine, ascorbic acid) or -mercaptoethanol in the isolation buffer helps to reduce browning [22,23]. Weishing et al (1995) reviewed the various methodologies on isolation and purification of DNA for DNA profiling [8]. Honeycutt et al. (1992) succeeded in the isolation of nuclear DNA in sugarcane by using modified CTAB method [24]. Further, Sul and Korban (1996) described a highly efficient method for isolating genomic DNA from leave tissue of in vitro raised
Table 1: DNA yields from fresh and dried leaves DNA (g/g of leaves)SE

plants of Malus domestica and Nicotiana tabaccum by using modified CTAB protocol which includes reduced centrifugation runtimes for both separation of two-phase layers and precipitation of genomic DNA and the addition of lithium chloride during extraction for restriction digestion analysis [25]. By using various methods described by Willmitzer and Wagner (1981), Dellaporta et al. (1983), Rogers and Bendich (1988), Doyle and Doyle (1990), Couch and Fritz (1990), Sul and Korban (1996) and Porbbski et al. (1997) indicated very low DNA yield and no PCR amplification reactions [10, 13, 25-28]. Our protocol involves two step use of chloroform initially because of precipitation of chlorophyll and proteins. The addition of 5M NaCl and ethanol help in precipitation of DNA. Fang et al. (1992) observed that the addition of high concentration of NaCl increased the solubility of polysaccharides in ethanol, effectively decreasing co-precipitation of the polysaccharides and DNA [29]. Lodhi et al. (1994) indicated that higher concentration of NaCl was more effective for removing polysaccharides in Vitis species [30]. Contamination with RNA occurs when isolating genomic DNA, copious amounts of cellular RNA precipitated concomitantly with DNA. Several reports have indicated that the presence of RNA can suppress PCR amplification and lead to nonreproducible and unreliable DNA amplification patterns in RAPD analysis [31, 32]. The addition of RNase reduces the amount of RNA present in the genomic DNA. The modified protocol described in this paper was used for isolation and purification of DNA. The result indicated in V. aconitifolia the quantity of the genomic DNA was about 42-44 gg 1 from fresh leaves and 51-53 gg 1from dried leaves. In V. trilobata the total DNA yield was about 46-48 gg 1 of fresh young leaf and 55-57 gg 1of dried leaves. The yield in both Vigna species is considerably higher than methods suggested by Dellaporta et al. (1983) and Doyle and Doyle (1990) [9, 13] (Table 1).

----------------------------------------------------------------------------------------------------------------------------------------------------------------------------Present Method --------------------------------------------Plant Species V. aconitifolia V. trilobata Fresh 431.2 472.1 Dry 521.2 562.6 Dellaporta et al., (1983) -------------------------------------------Fresh 222.0 212.3 Dry 262.2 241.8 Doyle and Doyle (1990) ---------------------------------------------Fresh 241.3 181.6 Dry 281.5 242.1

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Fig. 1: The intact double stranded Purified DNA. Lane 1, 2 and 3: from V. aconitifolia; Lane 4, 5 and 6: From V. trilobata; Lane 7: Hind III digested -DNA as marker

Fig. 3: RAPD profile of the DNA samples from amplified with primer P1 and P2. Lane 1, marker (EcoR I / Hind III double digest genome); lane 2 and 3 RAPD of V. aconitifolia ; lane 4 and 5 RAPD of V. trilobata The total genomic DNA isolated by methods described by Rogers and Bendich (1985) [10] and Dellaporta et al. (1983) [13] provided intact DNA but yield was low while Porebski et al. (1997) [28] methods produced large amounts of sheared DNA. On the contrary, DNA isolated by presently described method produced good quality and high quantity of intact DNA. The DNA was examined for intactness using the gel electrophoresis method. The intact double standard DNA forming a thick single band of high molecular weight confirmed the good quality of the extracted DNA (Fig 1). DNA purity is a concern in extraction procedures. DNA purity was determined from the A260/280 ratio, which averaged 1.03-1.14 in all samples indicating the absence of contaminants [33]. Restriction endonuclease digestion requires fairly clean and large quantities of DNA [34]. Random amplified polymorphic DNA and related techniques require less DNA, but purity is necessary to ensure repeatability and confidence [35-36]. We successfully digested 2 g of isolated DNA with 8 U of EcoR I and 8 U of Hind III. The EcoR I and Hind III digestion was complete (Fig.2).
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Fig. 2: Restriction digestion of genomic DNA. Lane 1: EcoR1 Digested genome marker; Lane 2: Hind III digested genome of V. aconitifolia; Lane 2: EcoR1 digested genome of V. aconitifolia; Lane 3: Hind III digested genome of V. trilobata; Lane 4: EcoR1 digested genome of V. trilobata.

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RAPD analysis of genomic DNA was performed according to Williams et al. (1990) [36] using 25 ng of genomic DNA, 0.2 mM dNTP, 25ng of random primers (P1 and P2), 2.5 mM MgCl2 and 1 U Taq DNA polymerase in a 25-L reaction volume. Good amplification was obtained with both random primers (Figures 3). There was no interference with PCR amplification reactions. The proposed protocol was found to have advantages for isolation of DNA from different species of Vigna leaves. The amount of DNA recovered per gram of leaf tissue material was sufficiently high enough for PCR amplification. This method described here is efficient for the isolation of purified DNA from legume plant like Vigna having high composition of polyphenols and polysaccharides. It will also help in other recalcitrant legume plant system. REFERENCES

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