Journal of Phytology 2011, 3(2): xx-xx ISSN: 2075-6240 www.scholarjournals.org www.journal-phytology.com
JP-Tissue Culture
Efficient DNA Isolation Method for Molecular Studies from Leaves and Roots of Rice (Oryza sativa) Mohiuddin Khan Warsi, Arif Tasleem Jan, Mudsser Azam, Swati Wanwari, Qazi Mohd. Rizwanul Haq* Department of Biosciences, Jamia Millia Islamia, New Delhi - 110025
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Article History Received Revised Accepted
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19-12-2010 03-03-2011 07-03-2011
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An efficient DNA isolation protocol specifically modified to get pure quality DNA required for the purpose of polymerase chain reaction amplification, Southern blot, RAPD, besides being necessary to study transcription factor genes during the drought/salt stress leaf of rice (Oryza sativa) has been reported in this paper. In comparison to other known protocols that require expensive equipment and chemical, this protocol seems more economical for getting pure DNA that in turn forms an essential part of routine work of any molecular biology lab where concentration is more towards quality rather than quantity of DNA. Rather than concentrating more on commercially available DNA isolation kits, this protocol is more economical in getting pure quality DNA, free from proteins and polysaccharide compounds. In our case, purity of isolated DNA checked by measuring absorbance (A260 /A280) was excellent as the ratio of A260 /A280 was 1.75 - 1.92, thereby suggesting that it does not need any additional purification before proceeding for molecular analysis of the isolated DNA samples. From the above study, it becomes quite clear that this protocol being simple and economical finds wide application in genomic study as quality forms an important factor for performing different gene expression studies through PCR.
Key Words: DNA, Oryza sativa, Gene expression, Transcription factor gene
Introduction Rice is one of the most important crops in the world as half of the world’s population depends on rice as a staple food [1]. Rice – a tropical C3 grass that evolved in semi-aquatic habitat carries an old portfolio of tolerances and susceptibilities to abiotic stresses as compared to other crops. Rice that normally thrives in waterlogged soils shows moderate tolerance to salinity. Rice being highly sensitive to drought and cold is generally grown in tropics during dry season where adequate irrigation is available, so that it may suffer low temperature at seeding and high temperature at flowering stage. DNA discrimination techniques have been applied to various aspects of plant breeding such as marker-assisted selection using DNA markers, cultivar identifications of the cultivars for the protection of the breeding rights and for the prevention from the contamination of undesirable cultivars, and the examination of the relationships between closely-related cultivar lines [2]. Molecular marker analysis using PCR based markers in genomic studies greatly enhances speed and efficacy of crop improvement through selection of desirable traits in a genotype [3, 4]. For these PCR based marker assisted selection, an excellent method for genomic DNA isolation in terms of quality and quantity is essential for a particular crop. Most important consideration in successful extraction of plant DNA is to avoid contamination of polysaccharides and polyphenols besides avoiding shearing of
DNA is required during the extraction procedure. Polysaccharide contamination is a common problem encountered in the DNA extraction higher plant as it acts inhibitorily in the PCR reaction [5]. With regard to study of transcription factor genes in rice (Pusa basmati-1), that regulate expression of stress inducible genes such as DREB2 and zinc finger like transcription factor gene, it is necessary that DNA should be unsheared. As the study of transcription factors require analysis of large number of samples, a simple but efficient and inexpensive method that yield high quality of DNA is desired. For study of transcription factors, a reliable DNA extraction method with less risk of DNA shearing during and post extraction as well as good yield of high quality DNA is required. Several DNA extraction procedures for isolating genomic DNA from various plant sources have been described, including salt extraction method, CTAB method and their modifications as reported by Zhou et al, 1993, Liu et al, 1995, and Huang et al, 2000 [6, 7, 8]. However, published protocols available in literature could not be successfully used for isolating high quality DNA from all the plants equally. As we were interested in amplifying transcription factor genes including DREB2 (Dehydration responsive element binding protein) gene, Zinc finger protein gene, MYB and NAC transcription factor gene, we focused on optimizing an efficient plant DNA extraction protocol without the above-mentioned
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limitations that can be used against large number of fresh as well as frozen plant samples belonging to different species, besides being efficient in terms of time and cost and also suitable for long-term storage of DNA samples. Here, in this paper we are reporting a reliable and cost-effective method that can be effectively used to achieve amplification of various transcription factors such as DREB2 gene, Zinc finger protein gene etc in rice during the salt stress condition in Pusa basmati-1.
(8) Dissolve the pellet in 50μl TE. Leave at room temperature for 5 min then spin down and store at -20oC. Results Purity of DNA Purity of DNA was checked by the mean of absorbance ratio A260/A280 and A260/A230 for protein and polyphenolics/polysaccharides compound, respectively (Fig 1). For DNA quantification absorbance was measured at 260nm. A DNA sample solution consist OD value 1 at 260nm contain 50μg of DNA per millilitre [9].
Materials and Methods Plant material Select similar size of seeds of Oryza sativa were sterilized and grown under control condition at 28o C day/ 25o C at night with a 12 hrs light/12hrs dark photoperiod. After 15 Days of germination seedlings were exposed to drought/salt/cold stress. Following stress induction, leaf and roots were separated and used for DNA isolation. Required solutions • Extraction buffer: 50mM Tris-HCl (pH-8.0), 500mM NaCl, 10mM EDTA (pH-8.0), 1% Sodium Dodecyl Sulphate (SDS), 1% β- Mercaptoethanol (Immediately add). • Phenol: Chloroform: Isoamyl Alcohol (25: 24: 1). • Chloroform: Phenol (24:1) • 80% Alcohol • TE buffer (pH-8)
Fig1: DNA extracted from root and leaf at different stress condition in which lane 1,2,6,7and 8 from drought and lane 3,4,9,10 and 11 from salt stress.
PCR amplification For PCR amplification, using 120 ng of DNA as a template, a reaction mixture cocktail containing 1x reaction buffer, 1.5mM MgCl2, 120ng DNA, 0.20mM dNTPs(each one), 0.5μM gene specific primer pairs of multiple stress associated zinc finger gene/ DREB gene and 2.5 units Taq DNA Polymerase (Fermatas) was prepared. The final volume of reaction mix of PCR was 50μl. The PCR was run for 30 cycles each of 30 sec denaturation at 95oC, 1min annealing at specific temperature for different primer, 1 min extension at 72oC in a techne thermo cycler (TC-312). DNA and amplification product were subjected to electrophoresis for desired time in 1% agarose gel at 60 volts in TAE buffer (pH 8.0). Gel were stained with ethidium bromide (0.5μg/ml), visualized under UV transilluminator and photographed using Geldoc (BioRad) (Fig 2 and Fig 3).
Protocol for DNA isolation from Rice leaves and roots. (1) Grind quickly 0.5gm fresh leaf and root in liquid nitrogen to make a fine powder with the help of pestle and mortar. We can use an alternative option in absence of liquid nitrogen, sample along with the pestle and mortar keep at -80o C for overnight freezing. (2) Dissolve it in Extraction buffer in proportion of 1:2 or 1:4. Vortex it for 5 minutes completely. (3) Centrifuge at 10,000-12,000 rpm for 5 minutes on 4o C temperature (equally weigh tube opposite to each other minimize shearing of DNA during centrifugation). Take supernatant and equal volume of Phenol: Chloroform: Isoamyl Alcohol (PCI) again centrifuge at 10,000 rpm for 5 minutes on 4o C. (4) Take aqueous layer (Supernatant) and again add equal amount of PCI and centrifuge at 10,000 rpm for 5 minutes on 4oC. Take out supernatant and add equal volume of Chloroform: Phenol. Centrifuge it at above mention condition. (5) Take out supernatant and add 1/10th volume of 3M Sodium acetate and 2.5 times chilled 100% alcohol. Keep it at -20oC for 45 minutes. (6) Centrifuge at 13,000 rpm for 30 minutes on 4oC temperature. Discard supernatant and DNA pellet wash with 80% ethanol at 5000rpm for 5 minutes. (7) Remove all ethanol carefully and to dry pellet in air.
Fig 2: Gel photograph showing amplification of (A) DREB2 (Lane2 and 7 showing maximum amplification at 5hrs and 8hrs while as remaining lanes 3-6 at 24 hrs salt stress condition and (B) Actin (as internal control)
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[2] Fukami, M., Y. Muramoto and K. Ohkoshi, 2008. Rapid and simple DNA extraction method from rice using a glass-fiber filter inserted pipette tip. Plant Biotech., 25: 493-496. [3] Pamidimarri, D.V.N.S., Meenakshi., R. Sarkar., G. Boricha and M.P. Reddy, 2009. A simple method for extraction of high quality genomic DNA from Jatropha curcas for genetic diversity and molecular marker studies. Indian J Biotech., 8: 187-192. [4] Chuan, S., C. Gang., R. Yu-chun., Z. Guang-heng., G. Zhen-yu, L. Jian., J. Pei-na, H. Jiang., G. Long-biao., Q. Qian and Z. Da-li, 2010. A simple method for preparation of rice genomic DNA. Rice Science. 17: 1-4. [5] Porebski, S., L.G. Bailey and B.R. Baum, 1997. Modification of CTAB DNA extraction protocol for plants containing high polysaccharide and polyphenol components. Plant Mol Biol Rep., 15: 8-15. [6] Zhu, H., F. Qu and L.H. Zhu, 1993. Isolation of genomic DNAs from plants, fungi and bacteria using benzyl chloride. Nucl Acids Res., 21: 5279–5280. [7] Liu, Y.G., N. Mitsukawa., T. Oosumi and R.F. Whittier, 1995. Efficient isolation and mapping of Arabidopsis thaliana T-DNA insert junctions by thermal asymmetric interlaced PCR. Plant J., 8: 457–463. [8] Huang, J., X. Ge and M. Sun, 2000. Modified CTAB protocol using a silica matrix for isolation of plant genomic DNA. Biotechniques., 28: 432-434. [9] Sambrook, J and D. W. Russel, 2001. In Molecular cloning: A laboratory manual. Cold Spring Harbor Laboratory Press, New York. [10] Hugo, R.P., V.R. Maria and H.V. Ruben, 1998. A simple method for isolating high yield and quality DNA from cotton (Gossypium hirsustum L.) leaves. Plant Mol Biol Rep., 16: 1-6. [11] Triboush, S.O., N.G. Danilenko and O.G. Davydenko, 1998. A method for isolation of chloroplast DNA and mitochondrial DNA from sunflower. Plant Mol Biol Rep., 16: 183-189. [12] Suman, P.S.K., K.S. Ajit., M.P. Darokar and K. Sushil, 1999. Rapid isolation of DNA from dry and fresh samples of plants producing large amounts of secondary metabolites and essential oils. Plant Mol Biol Rep., 17: 17. [13] Callahan, F.E and A.M. Mehta, 1991. Alternative approach for consistant yields of total genomic DNA from cotton (Gossypium hirsustum L.). Plant Mol Biol Rep., 9: 252-261. [14] Collins, G.G and R.H. Symons, 1992. Extraction of nuclear DNA from grape vine leaves by a modified procedure. Plant Mol Biol Rep., 10: 233-235. [15] Daboo, S.M., E.D.J. Mitchell and U. Melcher, 1993. A method for the isolation of nuclear DNA from cotton (Gossypium) leaves. Anal Biochem., 210: 34-38.
Fig3: Gel photograph showing amplification of zinc finger gene: from drought stress and (B) from cold stress plants (lane1-is control, Lane2-2hrs, Lane3-4hrs, Lane 4,5 and 6 from 8hrs and lane 7-9 from 24 hrs drought/salt stress plants).
Discussion This method is more efficient then the other DNA isolation methods [10]. It does not need ribonuclease and protenase in isolation step. This protocol is more economic than other traditional techniques [11].We can improve the quality of extracted DNA by changing the concentration of βmercaptoethanol and NaCl in extraction buffer. We can remove the polyphenols by using high levels of β-mercaptoethanol [12]. The purity of extracted DNA was excellent after this modification. The absorption ratio (A260/A280) of extracted DNA samples that range in between 1.8 -1.9 suggesting that the preparation was free from protein and polyphenols. DNA yield is important in stress related molecular work. On basis of this result we can say there is no need to go for purification before using it for molecular studies. Rice seed germination and stress treatment is a time consuming process. Stress is complex phenomenon that can occur at any point during crop production and for any length of time, affecting a large array of physiological, biochemical and molecular processes. These complexities, along with the uncertainty in stress timing, intensity, and duration, continuously possess a major challenge for agricultural crops. In this regard, extraction of DNA for studying various aspects of plants under different stress condition forms an important step. Using this method, we obtained a good amount of high quality of DNA, up to 529μg/g of fresh tissue. This yield is higher than other reported methods of Callahan and Mehta, 1991; Collins and Symons, 1992 and of Dabao et al., 1993 [13, 14, 15]. Acknowledgement Mohiuddin Khan Warsi would like to thank UGC (New Delhi India) for fellowship. One of the authors, Arif Tasleem Jan would like to thank Council for Scientific and Industrial Research, India for fellowship in terms of SRF. References [1] Lafitte, H.R., A. Ismail and J, Bennett, 2004. Abiotic stress tolerance in rice for Asia: Progress and the future. Proceedings of 4th International Crop Science Congress (www.cropscience.org.au).
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