Download Rapid Method for Extraction of Genomic DNA From Vitex negundo L.

Malaysian Journal of Science 29(1) : 37 - 40 (2010)
Rapid Method for Extraction of Genomic DNA From Vitex negundo L.
R. Mary Sujin1, A. John De Britto2*, R. Mahesh3 and K. Dharmar4
1234
Plant Molecular Biology Research Unit, St.Xavier'sCollege, (Autonomous) Palayamkottai - 627 002,
Tamilnadu, India.
*[email protected] (corresponding author ).Tel: 0091- 462 4264374, Fax: 0091- 462-2561765.
Received on 28 August 2009, accepted in revised form 16Th March 2010
ABSTRACT
DNA extracted from plants which contains rich amount of polyphenols and or
polysaccharides are often problematic when subjected when mature tissues are used for DNA extraction. In
order to overcome these problems, we develope the first reliable and efficient method for isolating vitex
negundo L. genomic DNA that is free from solubilising polysaccharides and polyphenols. This protocol
uses NaCl, PVP, mercaptoethanol, SDS and incubation at 68°C for 1 hour. Mild temperature conditions
during extraction and precipitation were also recognized as important parameters. The quantity of isolated
genomic DNA was confirmed by means of spectrophotometic analysis (260/280=1.6-1.8) routinely
yielding 250-500 ng/µl per gram of leaf material and it was proved through RAPD-PCR analysis.
(Keywords: DNA extraction, RAPD-PCR analysis, Vitex negundo L.)
achieved by using detergents such as sodium
dodecyl
sulphate
(SDS)
or
cetyltrimethylammonium bromide (CTAB). This
released DNA should be protected from
endogenous nuclease. The initial DNA extracts
often contain large amounts of RNA, proteins,
polysaccharides, tannins and pigments, which
may interfere with the extracted DNA and are
difficult to separate. Most proteins are removed
by means of denaturation and precipitation from
the extract with chloroform and /or phenol.
Polysaccharides contaminates are particularly
problematic [6].They can also coprecipitate with
DNA after alcohol addition during DNA
isolation to form highly viscous solutions [7].
INTRODUCTION
Vitex negundo L. (Local Name-Nochi) belongs
to the family Verbenaceae. It is a small tree
credited with innumerable medicinal activities
like anti-cancer, analgesic, anti-inflammatory,
anticonvulsant, antioxidant, bronchial relaxant,
hepatoprotective, etc. The ethanolic extract of
leaves has been found safe as LD50 dose (by oral
route) of it was recorded in non-toxic dose range
[1].
The roots, fruits, flowers, leaves and
bark of V. negundo species are used for
medicinal purposes like foot and mouth diseases,
impaction with fever [2]. Juice extracted from
100gm leaves is consumed to cure Dyspnoea
(Shwasam mutt) [3]. Leaves are smoked in
headache. Flowering tips are chewed for curing
ulcers. Twigs are used as toothbrush. Leaf paste
is applied for curing wounds [4].
Antioxidants are commonly used to
address problems related to phenolics. Examples
include the use of β-mecaptoethanol, ascorbic
acid, bovine serum albumin (BSA), Sodium
azide and polyvinylpyrrolidine (PVP) [8] [9].
Phenol extraction coupled with SDS is also
helpful. However SDS tends to produce low
DNA yields of plants rich in polyphenlics [10].
Isolation of plant nucleic acids for use
in PCR, RFLP, RAPD, AP-PCR, DAF and
genomic library construction is one of most
important and time-consuming steps. The degree
of purity and quantity varies among applications.
A good extraction procedure for the isolation of
DNA should yield adequate and intact DNA of
reasonable purity. Dangerous chemicals should
not be used, if possible, and the procedure should
be simple, inexpensive and quick [5].
The optimised protocol described here
is specifically designed to isolate genomic DNA
of Vitex negundo L. and amplification detecting
through RAPD-PCR techniques.
MATERIALS AND METHODS
Plant materials
The leaves sample of Vitex negundo L. were
used to determine the extracting sufficient
amount of DNA. The leaves were collected
during the summer season (April). The collected
The extraction process involves
breaking or digestion away cell walls to release
the cellular constituents. This is followed by
disruption of the cell membranes to release DNA
into the extraction buffer. This is normally
37
Malaysian Journal of Science 29(1) : 37 - 40 (2010)
leaves were placed in plastic bag and stored in a
deep freezer at -70°C.
Reagents and chemicals
•
•
•
•
•
An extraction buffer consisting of 2%
SDS, 800 mM NaCl, 25mM
Tris/HCl(pH 8.0), 25mM EDTA 2%
PVP and 0.8% β-mecaptoethanol was
prepared.
Chlorform-isoamylalcohol (24:1)
Wash solution: 15mM potassium acetate,
75% ethanol
TE buffer: 10mM Tris-HCl (pH 8), 1mM
EDTA (pH 8)
0.5V of isopropanol
DNA extraction protocol
•
•
•
•
•
•
•
•
•
Plant tissues (200 mg leaf) were quickly
frozen in liquid nitrogen, powdered
with motor and pestle and transferred
into 1000µl of extraction buffer.
The extract was slightly mixed and
incubated in a waterbath at 68°C for 1 hour.
Then 15mM potassium acetate was
added followed by 30 minutes
incubation on ice and centrifugation
(12000rpm, 10 minutes, 4°C).
The supernatant was transferred into a
new tube, mixed 0.5 vol isopropanol by
inverting the tubes three times and
incubated on ice for 10 minutes.
After another centrifugation (12000rpm,
10 minutes, 4°C) the supernatant was
discarded completely.
The pellet was dried under vaccum,
dissolved in 20 µl 1×TE and extracted
once with 500 µl of Chlorformisoamylalcohol (24:1).
After centrifugation (12000rpm, 10
minutes, 4°C) the aqueous phase was
transferred and nucleic acids were
precipitated
in
0.5V
of
isopropanol(20°C, 15 minutes).
During this time the tubes were gently
inverted at least ten times. Finally the
tubes centrifuged again (12000rpm, 10
minutes, 4°C)
The pellet was washed in 75% ethanol,
dried under vaccum and dissolved in 20
µl 1×TE.
Store the DNA at -2°C for long-term
storage. If needed, treat the DNA
solution with RNase before use.
38
DNA obtained with this technique
constantly gives a 260/280 absorbance ratio of
1.6-1.8 indicating high quality intact DNA [11].
All of these DNA were used for RAPD-PCR
analysis using the method [12]. Arbitrary
decamer primes successfully amplified DNA
fragments from Vitex negundo L.
RESULTS AND DISCUSSION
Medicinal plants have large amounts of
secondary metabolites. These secondary
metabolites make hindrance in DNA isolation
and isolated DNA is not suitable for PCR
amplification and restriction digestion. We
followed the protocol described by [13].
Midiprep method for the isolation of DNA from
plants with a high content of polyphenolics.
DNA extracted, however was very
viscous and could not be resolved on the agarose
gel. The other problems encountered were low
DNA yields and poor PCR amplification as well
as restriction digestion. We increased the
concentration of PVP, β-mercaptoethanol and
NaCl and incubation temperature from 65°C to
70°C temperature. These modifications helped in
the extraction of DNA of high purity and high
quantity. We also eliminated the use of phenol
for the purification of extracted DNA, which
makes our method less hazardous. We also used
2 µl of RNase per 20 µl DNA samples for
removal of RNA. The DNA yields and resolution
on agarose gel however, were much higher and
better from both fresh and dried root samples of
medicinal plants with the use of our protocol.
Sufficient amount of highly purified
clean genomic DNA was obtained when the
optimised protocol was described by us.
All of these DNAs were used for
RAPD-PCR analysis. Arbitrary decamer primers
successfully amplified DNA fragments from 5
accessions of Vitex negundo L. The DNA
sample was extracted using this procedure and
assayed for RAPD-PCR using the primer OPX17(CAGACAAGCC) by using a TGradient
thermal cycler. Each PCR 25 µl reaction mixture
consisted of 4.0 µl dNTPs, 0.3 µl MgCl2, 2.5 µl
PCR Buffer, 2.4 µl Primer, 0.5 µl
Taq
polymerase, 13.3µl
Sterile water, 2.0 µl
template DNA. After the initial denaturation
94°C for 5min, 35 PCR cycles were performed
with 35 sec at 93°C, 55 sec at 53°C and 45 sec at
72°C,. The DNA extracts as well as the
amplification products were run in 2% agarose
gels repectively, stained with ethidium bromide
and visualized under UV light.
Malaysian Journal of Science 29(1) : 37 - 40 (2010)
The different protocol are described
earlier for isolation of genomic DNA from
medicinal plants such as protocol for isolation of
genomic DNA from dry and fresh roots of
medicinal plants suitable for RAPD and
restriction digestion [14], DNA protocols for
plants [15], DNA isolation methods for
medicinal and aromatic plants [16] and
optimization of DNA isolation and PCR protocol
for RAPD analysis of selected medicinal and
aromatic plants of conservation concern from
Peninsular India [17].
Fig1. shows the results of PCR
amplification using the primer OPX 17 of Vitex
negundo L. This DNA preparation method
yielded a prdominance of high molecular wieight
DNA reliably produced PCR amplification
products. Considering all factors involved, this
method appears to be the most efficient, reliable
and labor-effective DNA isolation procedure
from plants containing a high content of
polyphenolics.
Figure 1: Showing the Genomic DNA and PCR- amplification product using RAPD OPX- 17 primer of
Vitex negundo L.
This method is a rapid, reliable and cost
efficient method for the isolation of the PCRquality DNA from the Vitex negundo L. The
DNA solutions are stable at 4°C for atleast two
months and can be kept frozen at -20°C for
longer storage. We anticipate that adoption of
this protocol among the vitex species will speed
genotyping, the mapping of mutants, transgenic
studies and other PCR based applications.
2.
Ambika Nag, Praveen Galav and
Kalewa, S.S. (2007). Indigenous animal
health care practices from Udaipur
District, Rajasthan. Indian Journal of
Traditional Knowledge. 6(4): 583-588.
3.
Arun Vijayan, Liju, V.B. Reena John,
J.V. Parthipan, B. and Renuka, C.
(2007). Traditional remedies of Kani
tribes of Kottoor Reserve forest,
Agasthyavanam, Thiruvananthapuram,
Kerala. Indian Journal of Traditional
Knowledge. 6(4): 589-594.
4.
Saroj Verma and Chauhan, N.S.
(2007). Indigenous medicinal plants
knowledge of Kumhar forest division
district Solan. Indian Journal of
Traditional Knowledge. 6 (3):494-497.
5.
Daneshwar Puchooa and Sher Ullah
Shahnoo Sulliman Khoyratty (2004).
ACKNOWLEDGMENTS
The authors thanking the International
Council of Medicinal Research, Government of
India, New Delhi (Ref: 59/12/2006/BMS/TRM
dt.) for financial support.
REFERENCES
1.
Vishal Tandon R. (2005). Medicinal
uses and biological activities of Vitex
negundo. Natural Product Radiance.
4:162-165.
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Malaysian Journal of Science 29(1) : 37 - 40 (2010)
Genomic DNA extraction from Victoria
amazonica. Plant Molecular Biology
Reporter. 22: 195a-195j.
and J. P. W. Young (eds.), Molecular
Techniques in Taxonomy. NATOASI
Series H, Cell Biology. 57:283-293.
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Scott, K.D. and Playford, J. (1996).
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rain forest plant species. Biotechnique.s
20:974-978.
7.
Do, N. and Adams, R.P.(1991). A
simple technique of removing plant
polysaccharides contaminants from
DNA. Biotechniques. 10:162-166.
16. Anna Maria Pirttilä, Merja Hirsikorpi,
Terttu Kämäräinen, Laura Jaakola and
Anja Hohtola (2001). DNA Isolation
Methods for Medicinal and Aromatic
Plants. Plant Molecular Biology
Reporter 19: 273a–f.
8.
Dawson, C.R. and Magee, R.J. (1995).
Plant tyrosinase (polyphenol ozidase)
In: Colowick SP and Kaplan NO (eds).
Methods in Enzymology. 2: 817-827.
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Clark, M.S. (1997). In: Plant Molecular
Biology-A
laboratory
Manual.
Springer-Verlog Berlin Heidelberg,
New York. 305-328.
17. Padmalatha, K. and Prasad, M.N.V.
(2006). Optimization of DNA isolation
and PCR protocol for RAPD analysis of
selected medicinal and aromatic plants
of
conservation
concern
from
Peninsular India. African Journal of
Biotechnology. 5 (3):230-234.
10. Rezaian, M.A. and Krake, L.R. (1987).
Nucleic acid extraction and virus
detection in grape vine. J Virol Meth.
17:277-285.
11. Sambrook, J. and Russel (2000).
Molecular Cloning: A laboratory
manual 3rd edn. Cold Spring Harbor
Laboratory Press. New York: 78-85.
12. Williams, J.G.K. Kubelik, A.R. Livak,
K.J. and Rafalski, J.A. (1990). DNA
polymorphisms amplified by arbitrary
primers are useful as genetic markers.
Nucleic Acids Research. 18: 6531 6535.
13. Uta Pich and Ingo Schubert (1993).
Midiprep method for isolation of DNA
from plants with a high content of
polyphenolics. Nucleic Acids Research.
21(14): 3328.
14. Salim Khan, M., Irfan Qureshi,
Kamaluddin, Tanweer Alam and Abdin,
M. Z. (2007). Protocol for isolation of
genomic DNA from dry and fresh roots
of medicinal plants suitable for RAPD
and restriction digestion. African
Journal of Biotechnology. 6 (3):175178.
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plants. in: G. Hewitt, A. W. B.Johnson,
40
Madras Agric. J., 96 (7-12): 293-295, December 2009
Detection of DNA Polymorphism by RAPD-PCR Fingerprint in
Plumbago zeylanica L. from Western Ghats
A. John De Britto*, R. Mahesh, R. Mary Sujin and K. Dharmar
PG & Research Department of Plant Biology and Biotechnology,
St.Xavier'sCollege (Autonomous), Palayamkottai-627 002
Studies were undertaken for detection of DNA polymorphism through Random Amplified
Polymorphic (RAPD) DNA fingerprint in Plumbago zeylanica L. collected from ten locations in
Western Ghats. A total of 27 DNA fragment (bands), ranging from 4 to 6 was observed. The
genetic distance between the population ranged from 0.1178 to 0.8109 and the genetic identity
ranged from 0.4444 to 0.8889. The overall observed and effective number of alleles is 0.3958
and 0.3599 respectively. Over all genetic diversity is 0 .1817 and percentage of polymorphic
loci was 81.48. The genetic similarity analysis was conducted on the basis of presence or
absence of bands which revealed a wide range of variability within the populations. The
cluster analysis clearly showed that there was high degree of diversity within the population.
The closest genetic distance existed within population. Thus these RAPD markers have the
potential for conservation of identified clones and characterization of genetic relatedness
among the population.
Key words: Gene diversity, RAPD-PCR analysis, Plumbago zeylanica
Plumbago zeylanica L. is an important
medicinal herb. It has high medicinal properties. It
belongs to the family Plumbaginaceae. Local name:
Vellai sidhirai mulam and Kotivelli. Plumbago
zeylanica, a rambling subscandent perennial herb
or under shrub with green branches, stems
somewhat cylindrical, spreading, terate, striate,
glabous. Leaves opposite/alternate, ovate or
oblong, petiole narrow, amplexicaul at the base and
often dialted into stipule like auricles. Flowers white,
in axillary and terminal elongated spikes, bisexual.
Calyx densely covered with stalked, sticky glands,
corolla white, very slender, tubular, stamens 5, free,
ovary superior, 5-gonous, one celled, and ovule
single and basal (John De Britto and Mahesh 2007).
The fruit, leaves, root of Plumbago zeylanica L.
have high medicinal and economic value
(Rajendran and Agarwal, 2007). It is used against
constipation (Pande et al, 2007). The root of
Plumbago zeylanica has numerous therapeutic
uses. The root is known to be abortifacient and to
have vesicant properties. It is used as appetizer and
expectorant and used for dysentery, diarrhoea,
diuretic and peptic ulcers. The root paste is applied
externally for filarial diseases. It is used externally
for early maturation, rupture and healing of abscess.
The root powder taken orally along with honey
gradually reduces hypercholostraemia and
improves blood formation (anaemia). It is used to
reduce obesity, vitiligo, splenomegaly, hepatomegaly
and ascitis. It is also used to relieve coryza (running
nose), hoarseness of voice and sore throat. It is
*Corresponding author email: [email protected]
used in the form of local applications for leucoderma,
scabies, psoriasis, symptoms of leprosy and allied
skin diseases. The decoction of the root is useful in
checking and preventing spermatorrhoea (Madhava
Chetty et al, 2006).
The conservation of plant population and
species is mostly concerned with the number of
individuals present in the populations in order to
assess factors such as genetic drift, inbreeding
depression and lack of mates in self compatible
species (Barrett and Kohn, 1991).
Molecular techniques help researches not only
to identify the genotypes but also in assessing and
exploiting genetic variability through molecular
markers. Random Amplified Polymorphic DNA
(RAPD) analysis has proved for estimating genetic
diversity particularly to assist in the conservation of
rare species and plant genetic resources. RAPD
analysis in particular has proven to be a rapid and
efficient means of genome mapping and is well
suited for the genetic resource characterization
(Williams et al, 1990). The DNA fingerprinting
generated by the polymerase chain reaction using
arbitrary primers has provided a new tool for DNA
polymorphism in number of herb species
(Padmalatha and Prasad, 2006).
In the present study the detection of DNA
polymorphism in P. zeylanica L. collected from ten
locations of Western Ghats has been carried out
using RAPD markers.
294
Materials and Methods
The extensive field survey was carried throughout
Western Ghats and ten accession of P. zeylanica L.
were collected from Kannanur, Kalakad, Kothyar,
Chunkankadai, Maruthamalai, Thiruparapu,
Kollimalai, Aralvoimozhi, Karayar and Courtallum.
Five plant samples from each location were
collected. The plant materials were transferred to
plastic bags for transport from field to laboratory.
The samples were maintained in deep freezer at
70° C till the analysis was done. Young leaf tissues
were used for extracting total genomic DNA, following
protocol suggested by (Uta Pich and Ingo Schubert
(1993).
The amount of DNA and its quality were
assessed by UV spectrophotometer. The DNA was
pure enough (OD260/280 =1.68), (Sambrook and
Russel, 2000) for RAPD- PCR analysis (Williams et
al., 1990).
Earlier, ten primers were tested and five primers
OPX 03 (GGAGGGTGTT), OPX 04 (CCGCTACCGA),
OPX06 (AGGGGTCTTG), OPX12 (TCGCCAGCCA),
OPX19 (TCTGTGCTGG) which produced
reproducible bands and were selected. The
experiments were repeated three times and
confirmed the reproducibility of bands. PCR
reactions were carried out in a total volume of 20 µl
at a final concentration of 1 mM MgCl2, 2 mM dNTP,
Taq DNA polymerase enzyme (1ul/20 µl), with
approximately 200 ng DNA as a template and a single
random primer (0.2 mM). The PCR cycles were as
follows: 94°C for 2 min, 94°C for 15 sec, 35°C for 15
sec, 72°C for 30 sec which were repeated in 35
cycles followed by 5 min-extension at 72°C. 8 µl of
PCR product was subjected to electrophoresis at
80 V using 1.5 % agarose gel stained with ethidium
bromide.The bands were viewed under UV
transilluminator and photographed with gel
documentation system Alpha Imager 1200.
Based on the primary data (presence or absence
of bands), pair wise genetic distance between
samples was calculated using POP GENE package
version 1.31. (Yeh et al., 1999).
Results and Discussion
Analysis of ten accession of P. zeylanica L.
revealed 22 polymorphic loci. Five primers
generated reproducible, informative and easily
scorable RAPD profiles. A total of 27 bands were
scored for the 5 RAPD primers out of which 115
bands polymorphic with number of bands ranging
from 4 to 6 (Plate 1). The same type of bands
occurred at different frequencies in all populations.
There were many additional bands neglected which
were not reproducible. The percentage of
polymorphic loci was 81.48. The genetic distance
between the population ranged from 0.1178 to
0.8109 and the genetic identity ranged from 0.4444
to 0.8889 which are shown in Table 1. As it was
Plate 1. Plumbago zeylanica L.
observed in the Popgene software results, the
overall observed and effective number of alleles was
1.8 and 1.3 respectively and overall genetic diversity
was 0 .2719. The obtained result was analyzed in
the POP GENE package 1.31 and the dendrogram
obtained clearly indicates three clusters (Nei, 1978)
(Fig 1).
Population 5 form a separate clade. Populations
1,6,2,4 and 7 form one clade and populations 3,9,8
and 10 form another clade. Three groups are
distinctly formed in the first clade (populations 1
and 6,2 and 4 and 7). In the second clade again
three groups are formed (populations 3 and 9, 8
and 10) (Fig 1).
The utility of RAPD markers in estimating genetic
divergence has been demonsrated in several
studies. The similar study was done in RAPD in in
vitro regenerated leaf explants of Plumbago
zeylanica L. (Rout, 2002). Genetic diversity analysis
in Rauvolfia serpentina and Rauvolfia tetraphylla L,
using RAPD Markers. (Padmalatha and Prasad
2007; Padmalatha and Prasad, 2006). Recently
studies on molecular analysis in Urginea indica
Kunth collected from different location of Karnataka
was reported. (Harini et al., 2008).
The clustering results of different accessions
suggest that Plumbago zeylanica L. undergoes
major part of genetic variation by environmental
factors. Genetic diversity refers to the variation at the
level of individual genes (polymorphisms), and
provides a mechanism for populations to adapt to
their ever-changing environment. The genetic
variability in P. zeylanica L. may be partly explained
295
Table 1. Nei’s Original Measures of Genetic Identity and Genetic distance
pop ID
1
2
3
4
5
6
7
8
9
10
1
****
0.8519
0.7407
0.8148
0.5556
0.8519
0.8519
0.7778
0.7037
0.6667
2
0.1603
****
0.6667
0.8889
0.6296
0.7778
0.77778
0.7037
0.7778
0.5926
3
0.3001
0.4055
****
0.5556
0.4444
0.6667
0.6667
0.7407
0.8148
0.7778
4
0.2048
0.1178
0.5878
****
0.7407
0.7407
0.8889
0.6667
0.6667
0.6296
5
0.5878
0.4626
0.8109
0.3001
****
0.4815
0.6296
0.5556
0.4815
0.5185
6
0.1603
0.2513
0.4055
0.3001
0.7309
****
0.7778
0.7778
0.7037
0.5185
7
0.1603
0.2513
0.4055
0.1178
0.4626
0.2513
****
0.7778
0.7037
0.7407
8
0.2513
0.3514
0.3001
0.4055
0.5878
0.2513
0.2513
****
0.7778
0.6667
9
0.3514
0.2513
0.2048
0.4055
0.7309
0.3514
0.3514
0.2513
****
0.6667
John De Britto, A. and Mahesh, R. 2007. Evolutionary
medicine of kani tribal's botanical knowledge in
Agasthiayamalai Biosphere Reserve, South-India.
Ethnobotanical Leaflets, 11: 280-290.
Madhava Chetty, K., Sivaji, K., Sudarsanam, G. and Hindu
Sekar, P. 2006. Pharmaceutical studies and
therapeutic uses of Plumbago Zeylanica L. roots.
Ethnobotanical Leaflets, 10: 294-304.
Nei, 1978. Estimation of average heterozygosity and genetic
distance from a small number of individuals. Genetics,
89:583.
Figure 1. Dendrogram Based Nei’s Genetic
distance
as a result of abiotic- geographical, hydrographical
connections, climatic differentiations - annual
rainfall, temperature, pH of the soil, humidity etc.,
and also the various biotic factors.
Genetic variation in a population is measured
by the heterozygosity or the degree of polymorphism.
For the conservation of a species, genetic variability
is of the utmost importance to preserve. Genetic
variability among all species is important to maintain
since it represents the 'blue print' for all of the living
things on earth.
It is desired to maximize the preservation of alleles.
Geographical, climatic or reproductive variables
explain the partitioning of the diversity observed which
may aid in improving the strategies for maximizing the
efficiency of germplasm collection and preservation.
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polyphenolics. Nucleic Acids Research. 21: 3328.
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Received: December 02, 2008; Revised: September 20, 2009; Accepted: November 20, 2009