versión On-line ISSN 0717-3458
Electron. J. Biotechnol. v.10 n.3 Valparaíso jul. 2007
A simple method for isolation of genomic DNA from fresh and dry leaves of Terminalia arjuna (Roxb.) Wight and Arnot
Vishal P. Deshmukh
Prashant V. Thakare*
Uddhav S. Chaudhari
Prashant A. Gawande
Financial support: Late Mukand Narhari Kale Research Scholarship Sant Gadge Baba Amravati University, Amravati.
Keywords: Ayurveda, DNA extraction, medicinal plant, Terminalia species.
Current protocols for isolation of genomic DNA from Terminalia arjuna have their own limitations due to the presence of high content of gummy polysaccharides and polyphenols. DNA isolated by these protocols is contaminated with a yellowish, sticky and viscous matrix. In our modified DNA isolation method polysaccharides and polyphenols are removed prior to the precipitation of the DNA. Then the genomic DNA was precipitated using isopropanol. This protocol yielded a high molecular weight DNA isolated from fresh as well as dry leaves of T. arjuna, which was free from contamination and colour. Isolated DNA can be used for restriction digestion and PCR amplification. The main objective of the present protocol is to provide a simple method of isolation of DNA, using in house prepared reagents.
Terminalia arjuna (Roxb.) Wight and Arnot (family Combretaceae) is a large tree distributed throughout
Polyphenolic contents were reported from T. arjuna by Bajpai et al. (2005). A little is known about the molecular biology of T. arjuna (Bharani et al. 2002), due to the presence of polyphenolic and polysaccharide compounds, which acts as inhibitors during isolation of DNA. During the isolation of DNA from perennial plant tissue like leaves of T. arjuna, these inhibitory substances get precipitated along with the DNA, thus deteriorating the quality and yield of the DNA. To solve this problem, we tried several protocols, which were reported previously along with various modifications from both fresh and dry leaves, but none yielded DNA free from polysaccharides and polyphenols. These situations required the development of a new protocol for the isolation of genomic DNA of high purity from T. arjuna.
We describe a simple DNA isolation protocol that yields high quality genomic DNA from fresh as well as dry leaves of T. arjuna. The isolated DNA has proved amenable to polymerase chain reaction (PCR) amplification and restriction digestion. The proposed method makes use of in-house prepared and readily available reagents and thus provides an alternative to the use of commercial DNA isolation kits. This protocol is applicable to other similar species of family Combretaceae e.g. Terminalia bellerica, Terminalia chebula and Terminalia tomentosa.
Juvenile leaves of T. arjuna, T. bellerica, T. chebula and T. tomentosa were collected from different locations of Melghat Tiger Reserve, Maharashtra, India (Latitudes 21º 15'N and 21º 45'N, Longitudes 76º 57'E and 77º 33'E and altitude
Genomic DNA (2-4 µg) was digested for 1 hr with 5 to 10 U of restriction enzyme under optimal temperature and buffer, following manufacturers recommendations (Fermentas, USA). The digested DNA fragments were fractionated on 0.7% agarose at 5 V/cm.
PCR was carried out in 25 µl volume reaction mixture. The reaction mixture contained 25 ng of DNA, 2.5 U Taq DNA polymerase enzyme (Fermentas, USA), 100 mM each dNTP,
The secondary metabolites produced by some plants possess important medicinal properties and are used in food, pharmaceutical, cosmetics and pesticide industries (Khanuja et al. 1999). T. arjuna contains ellagic acid, gallic acid, arjunic acid, arjungenin and their glucosides, arjunetin and arjunglucoside I (Bajpai et al. 2005; Pawar and Bhutani, 2005). The isolation of genomic DNA from perennial plants like T. arjuna is difficult due to presence of polyphenols and polysaccharides. During isolation procedure polysaccharides are found to form complexes with nucleic acids forming a gelatinous mass, thereby physically inhibiting the DNA from the action of DNA modifying enzymes e.g. restriction enzymes, DNA polymerase, ligase, etc. (Sharma et al. 2002). The polyphenols isolated along with DNA from T. arjuna are converted to several products reacting with proteins, and bring about their oxidation (Loomis, 1974). For the isolation of DNA from species like T. arjuna and T. tomentosa, we tried several published protocols like CTAB based (Doyle and Doyle, 1990), high salt and PVP (Porebski et al. 1997) high salt and sarcosyl (Sharma et al. 2002), combination of CTAB and SDS (Keb-llanes et al. 2002), using glucose as a reducing agent in standard CTAB protocol (Permingeat et al. 1998), and protocol for other species of Terminalia (Warude et al. 2003). The DNA isolated by above-mentioned methods was sticky, viscous and colored inhibiting the activity of DNA modifying enzymes. To overcome the problem of contaminant compounds, we tried modified protocols with higher concentration of CTAB (Khanuja et al. 1999). High ionic strength of CTAB forms complexes with proteins and most of the acidic polysaccharides (Jones and Walker, 1963); whereas high concentration of NaCl helps in the removal of polysaccharides (Aljanabi et al. 1999). In our study, we used the different concentrations of CTAB and PVP. However, in all the cases, low concentrations of polyphenols and polysaccharides were present at the end (data not shown). In our protocol, the polyphenols find their way in DNA preparation during the liquid nitrogen homogenization process. We crushed leaves in liquid nitrogen and removed the polyphenols by repeatedly washing 4-5 times using PVP and β-mercaptoethanol. The polysaccharides were removed using extraction buffer containing high NaCl concentration. Current protocol yields DNA of high purity and free from polyphenols and polysaccharides from fresh as well as dry leaves of T. arjuna, and other Terminalia species (Figure 1). The purity of the DNA sample was confirmed through its A260/A280 ratio (1.8) and digestion with restriction enzyme
In conclusion, the current method is simple and reliable for the isolation of genomic DNA from fresh and dry leaves of T. arjuna, which is known to be one of the complicated species for the isolation of DNA, due to the presence of a high percentage of secondary metabolites. This method was successfully applied for the isolation of DNA from other species of Terminalia like T. bellerica, T. chebula, and T. tomentosa.
We would like to thank anonymous reviewers for helpful comments. We are thankful to Prof. M.K.Rai, Head, Department of Biotechnology, SGB Amravti University for valuable suggestions and guidance.
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