Conservation of sandalwood genetic resources is mainly concerned with maintaining all the genetic variations contained within and among carefully selected target species. Hence DNA based markers RAPD which are excellent tools for studying diversity at the DNA level and assist in elimination of duplicates in germplasm and discriminating cultivars are widely used. Where we present the optimization of DNA isolation and PCR condition for RAPD analysis of selected Santalum album genotypes collected from Tamil Nadu, Karnataka and Andhra Pradesh. It is of great economic importance because of its fragrant heartwood and oil. (Rogers, 1996) India accounts for nearly 99 percent of sandal oil producer in the world. Sandalwood occupies an important place in the ecological, cultural and spiritual heritage of India. Extraction of high quality DNA from sandalwood leaves is a difficult task due to their rigid cell wall which is composed of complex carbohydrates. Sandalwood leaves are also rich in secondary metabolites, such as polyphenols, tannins and polysaccharides, which pose major problem in DNA purification, as they are difficult to separate from DNA. Many of the initial problems encountered in the extraction of high quality DNA have been attributed to these contaminants (Murray and Thompson,1980). A modified CTAB method DNA extraction (Probeski et al.,1997) was followed which is relatively quick, inexpensive and consistent for extraction of DNA.
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Keywords : DNA Isolation, Sandalwood, PCR Amplification, RAPD Analysis, DNA markers
Santalum album is of great economic importance because of its fragrance heartwood and oil. Which have high value in the cosmetic, perfumery and handicraft industries. Santalum album leaves contain high amount of polyphenols, tannins and polysaccharides. Problems encounter in the isolation and purification of DNA because of degradation of DNA due to endonucleases, highly viscous polysaccharides, inhibitor compounds like polyphenols and other secondary metabolites which directly or indirectly interference with enzymatic reaction. These contaminant causes many problems in PCR amplification, interfere with DNA amplification involving random primers and result in improper priming of DNA template during thermal cycle sequencing Pikkart et al10
Various protocols for DNA extraction have been successfully applied for this we used four methods 1 and 2 method using CTAB 3 and 4 methods using SDS methods. These are compared for isolation of DNA from sandalwood leaves. The protocol involving SDS was uniformly unsuccessful in attempts to extract high quality DNA. The main obstacle was difficulty in re dissolving Iso propanol precipitated DNA in TE buffer.
Optimization of PCR enzymatic method of making multiple copies of predicted segment of DNA. It commence with denaturation step of the DNA followed by primer annealing temperature, final extension and components in PCR mixture influence the final product. Hence these conditions have to be optimized in order to produce informative and reproducible finger prints.
MATERIALS AND METHODS
Material from different clones of Santalum album was obtained from germplasm block maintained by Institute of Wood Science and Technology (IWST), Bangalore, were sandalwood genotypes procured from different geographical regions of India are conserved. The present study was carried out on a set of 30 randomly selected clones, collected from different states namely, Karnataka (12), Tamil Nadu (14), Kerala (3) and Andhra Pradesh (1). Approximately 8-10g of dried leaves were powdered by using "Mixer" for 45-60 seconds and sieved through a 60 mm-mesh sieve to obtain a fine powder. The required quantity of powdered leaf sample was weighed and used for DNA extraction.
The extraction buffer consisting of 6% (w/v) CTAB, 5M NaCl, 1M Tris base ( pH 8 ) 0.5 M EDTA ( pH 8 ); chloroform: Isoamyl alcohol 24:1 v/v solution, 1% Î²-Mercaptoethanol, 3M Sodium acetate are prepared.
Recently matured leaves from 3rd to 5th node were collected before blooming. Processing of too young leaves resulted in charring of tissue while drying14. Leaves are dried in hot air oven at 40°C for 24 hrs. Dried leaves were powdered and sieved through 60mm mesh to obtain fine powder. Hence, four different genomic DNA purification protocols outlined by different workers with minor modification were evaluated to isolate high molecular weight DNA from sandalwood leaves.
The leaf sample for DNA extraction was prepared by following Tai and Tanksley method15 with minor modifications. They were wiped and dried at 40Ëš C for 24 hours. The dried leaves were then sealed in plastic bags and used later. Approximately 8-10g of dried leaves were powdered by using "Kanchan Mixer" for 45-60 seconds and sieved through a 60 mm-mesh sieve to obtain a fine powder. The required quantity of powdered leaf sample was weighed and used for DNA extraction.
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The protocol outlined by Porebski et al11 was followed with minor alterations. 500 mg of the leaf powder with 20 ml extraction buffer (20 mM Ethylene Diamine Tetra Acetic acid pH 8.0, 100 mM Tris-Base pH 8.0, 1.4 M NaCl, 3 % CTAB and 2% Poly Vinyl Pyrolidone with 1 % Î²-mercaptoethanol) was incubated on a water bath at 65Â° C for one hour with intermittent shaking. The mixture was then cooled on ice bath and 10 ml of cold chloroform: isoamylalcohol (24:1 v/v) was added and the contents were mixed well by inverting the tube and spun at 9000 rpm for 20 minutes. The supernatant was transferred to a fresh tube and this step was repeated four times till clear supernatant was obtained. To the aqueous phase, half the volume of 5 M NaCl was added and gently mixed followed by addition of one volume of cold propanol to precipitate the DNA. The solution was kept at 4Â° C over night to accelerate the precipitation of DNA. The mixture was spun at 10,000 rpm for 25 minutes to pellet the DNA. The pellet was washed with 70 per cent ethanol and dried in a vacuum drier for one hour. The pellet was resuspended in 300 Î¼l of TE buffer (10 mM Tris HCI and 1 mM EDTA pH 8.0) and the samples were stored in eppendorf tube at -40Â° C until further analysis.
Collection of recently matured leaves of Sanatalum album and drying them at 40Â° C for 24 hours was found optimum for powdering and further processing for DNA extraction (Fig 4a). Prolonged drying beyond 24 hours was found detrimental for the quality of DNA. Use of high salt concentrations (3% CTAB, 1.4M NaCl), 2 per cent PVP, 1 per cent b-mercaptoethanol in 20 ml extraction buffer for 400 mg of dried leaf powder and an extended RNAse treatment followed by phenol-chloroform (1:1) extraction resulted in high amounts of good quality DNA suitable for RAPD analysis. The quality of DNA isolated by method 2 was found good and complete cleavage of DNA by HindIII was observed, where as digestion of DNA was incomplete, partial for other methods (Fig. 4b). The OD 260/280 ratios ranged from 1.40 to 1.85 (Table 3). The success of different protocols was evaluated based on the usefulness of DNA isolated for PCR amplification.
DNA purification and assessment
Since the DNA isolated by the above methods may contain considerable amount of RNA, polysaccharides, phenolics and other impurities, the extract was subjected to further purification as follows To remove RNA, each sample was incubated with 3 g/ml RNAse on a water bath at 37Â° C for over night. The proteins were removed by mixing the solution with equal volume of phenol, then with phenol: chloroform (1:1 v/v) and finally with chloroform (spun at 10,000 rpm for 10-15 minutes). Two volume of cold ethanol was added to precipitate DNA. The precipitation was improved by keeping at -20Â° C for 1 hour. Then spun at 12,000 rpm for 20 minutes to pellet the DNA. The pellet was washed with 70 per cent ethanol and dried in a vacuum drier for one hour. The pellet was dissolved in 300 Î¼l of TE buffer and to it l/10th volume of 3 M sodium acetate was added and left overnight. The supernatant was collected after 24 hours by spinning at 12,000 rpm for 20 minutes. The DNA was quantified by using "ELICO Double beam Spectrophotometer" and verified by electrophoresis on 0.8 % agarose gel (Bangalore Genie) based on the intensities of band when compared with the lambada DNA marker used to determine the concentration. The purity of sandalwood genomic DNA was evaluated by measuring absorbance (A260 nm/A280 nm ratio) with a Double Beam UV spectrophotometer. Pure DNA preparations show a ratio between 1.8 to 2.0.
Optimization of RAPD reaction
Polymerase chain reaction is powerful technology used in many areas of molecular biology which allows in vitro amplification of specific DNA sequence7. DNA extracted from various Santalum album species, oligonucliotide primers from a to z series (Operon Technologies Inc, USA) were used for amplification and to standardize the PCR conditions.
The reactions were carried out in DNA thermo cycler (MJ Research Inc, USA). The PCR reactions were carried out in a final volume of 25 Âµl reaction mixture containing 25 ng template DNA 2.5 Âµl 10x buffer, 4.3 Âµl of 1.25mM dNTPs, 1 unit of Taq DNA polymerase, 3mM Mgcl2 and 5 pico moles primer (Integrated DNA technologies Inc). The PCR reaction was evaluated for 30, 40 and 45 cycles. The PCR mixture was overlaid with hot burn cap. Amplification was achieved in a MJ Research Thermalcycler (PTC-100) programmed for initial denaturation at 95Â°C for four minutes, followed by 45 cycles; each cycle consisting of denaturation at 94Â°C for one minute, primer annealing at 35Â°C for 2 minutes, primer extension at 72°C for 2 minutes, and a final extension of 10 minutes at 72Â°C. The PCR reactions were repeated three times
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Amplification product were resolved by electrophoresis in 1.2 percent agarose gel containg ethidium bromide (0.5Âµg/ml) using 1x TBE buffer wells were loaded with 25 Âµl of reaction mixture mixed with 5 Âµl of loading buffer. Electrophoresis was run at constant voltage of 60 volts for 5 to 6 hrs in 1x TBE buffer after that photographed under UV light by using Total Lab Gel documentation system.
The purity of the isolated DNA of Santalum album checked using double beam spectrophotometer to check the quality as it showed a reading between 1.8 to 2.0, after calculating the 260/280nm absorbance. The DNA yields obtained were ranged from the 1 to 2 Âµg/Âµl. The size, purity and integrity of DNA isolated were determined by agarose gel (0.8%) electrophoresis using Î» phage DNA cleaved with HindIII as a size standard. The quality of DNA obtained by different methods was compared by restricting with 2 units of HindIII restriction enzyme per microgram of DNA. Reactions were incubated at 37°C for overnight and then subjected to agarose gel electrophoresis, which was carried out in a horizontal gel system on 0.8 per cent with 1 X TBE buffer at constant voltage of 60v for 4 to 6 hours and staining the gel with ethidium bromide (0.5 Âµg/ml). The size of the amplified fragment 100bp to 4kbp.
First and foremost my thanks must go to my supervisor Dr. R. Nelson M.sc., Ph.D. for his guidance and great support throughout my studies. I would also extend my thanks to the A. Prasad Babu, DRR, Hyderabad for his valuable suggestions. And thanks to Institute of Wood Science and Technology, Research Divisions, Bangalore-560 003, India. For their kind help in sample collection.
Comparison of DNA yields from sandalwood leaf tissue using different methods of DNA isolation.
Dry weight of leaf tissue
DNA yield (mg/g dry tissue)*
OD ratio 260/280
* Average of 7 replications,
+ Poor amplification,
- No amplification
++ Good amplification,
The purity of DNA obtained from various clones varied from 1.70 to 2.01 (Table 4) indicating that the DNA was relatively of high purity and was suitable for PCR amplification.
Table 4.Absorbance and yield ratios of DNA from genotypes obtained after purification
(Î¼g/g dry wt.)
T15 Theosophical socity, Madras
T25 Thombakal RF,shanimadu, Harur, Chitteri, Dharmapuri
K3 Hallihatti,Rani bennur, Gadag
K25 Kuderekonda ayanur Shimoga
KL2 Anackelpety maryur Munnar
AP4 Nehru zoo Hyderabad
Agarose(0.8%) gel electrophoresis of sandalwood genomic DNA isolated by different methods
1 2 3 4 5 6 7 8 9 10
Lanes 1&2: DNA obtained by method 1 (Doyle and Doyle, 1987)
Lanes 3&4: DNA obtained by method 2 (Porebski et al., 1997)
Lanes 5&6: DNA obtained by method 3 (Edwards et al., 1991)
Lanes 7&8: DNA obtained by method 4 (Do and Adams, 1991)
Lanes 9&10: DNA obtained by method 2 (Porebski et al., 1997)
Agarose (0.8%) gel showing HindIII digestion of sandalwood genomic DNA Isolated by different methods
M 1 2 3 4
Lane M: l DNA/HindIII digest marker
Lanes 1: DNA obtained by method 1 (Doyle and Doyle, 1987)
Lanes 2: DNA obtained by method 2 (Porebski et al., 1997)
Lanes 3: DNA obtained by method 3 (Edwards et al., 1991)
Lanes 4: DNA obtained by method 4 (Do and Adams, 1991
PCR Optimum concentration and conditions for RAPD analysis
Hot start (95Â°C)
Number of cycles
2min, 3min, 4min, 5min
30sec, 1min, 1.5min
5 pico moles