A simple method for isolation of genomic DNA from fresh and dry leaves of Terminalia arjuna (Roxb.) Wight and Arnot
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.
Wight and Arnot (family Combretaceae) is a large tree distributed
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.
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.
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,
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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