Indian Journal of Biotechnology
Vol 11, January 2012, pp 67-71
An improved method of DNA isolation from polysaccharide rich leaves of
Boswellia serrata Roxb.
P Sharma and S D Purohit*
Plant Biotechnology Laboratory, Department of Botany, Mohanlal Sukhadia University, Udaipur 313 001, India
Received 29 September 2010; revised 20 July 2011; accepted 8 August 2011
A quick, simple and improved DNA extraction method from polysaccharide rich leaves of Boswellia serrata Roxb. has
been developed. Different methods involving use of CTAB and SDS with or without modification were used. A CTAB
based extraction method which uses diatomite to remove polysaccharides, such as, gums and resin, proved to be the best.
This method allowed recovery of good quality DNA in sufficient quantity, suitable for complete digestion by restriction
endonucleases and amplifiable in polymerase chain reaction as compared to other methods.
Keywords: Boswellia serrata, CTAB, diatomite, DNA extraction, PVP
Introduction
For molecular studies, a paramount requirement is
the isolation of DNA of sufficient mol wt and purity
suitable for restriction analysis, cloning and selective
sequence amplification. Plant DNA is generally
extracted by procedures derived from the hot CTAB
and SDS methods of Saghai-Maroof et al1 and
Dellaporta et al2, respectively. However, extraction of
intact, high quality DNA is challenging when working
with plant tissues rich in resins, gums, polyphenols,
polysaccharides and tannins3. For such taxa, isolation
of DNA from the CTAB and SDS complex gives a
product of inadequate purity, generally where
extraction of DNA is hindered by polyphenol and
their quinone oxidation products and by carbohydrate
polymers4,5. Several modifications of the original
procedures have been reported for the isolation of
DNA from plants with high polysaccharides and
polyphenols compounds6-8.
Boswellia serrata Roxb. (Family: Burseraceae),
commonly known as “Salar”or “Salai”, is a principal
tree of Aravallis in Rajasthan, India. It is of immense
medicinal importance. The tree is an important source of
gum-resin called “salai guggul” or “Indian olibanum”
obtained from the bark, which is extensively used in
pharmaceutical formulations for relieving pains and
aches,
particularly
associated
with
arthritis.
Over-exploitation of B. serrata from its natural habitats
has resulted into its depleting populations. Therefore,
conservation and sustainable utilization of this plant
species have become a serious concern for conservation
biologists, foresters and small industrialists. One of the
major constraints for its management is lack of any
molecular data on its genetic diversity in nature. A
pre-requisite for any molecular studies is the ability to
isolate DNA. B. serrata leaves contain high
polysaccharide contents, gums and resins which make
the DNA extraction difficult through conventional
methods. The present investigation was, therefore,
undertaken to test the suitability of available methods for
DNA isolation and make modifications, wherever
necessary, to get genomic DNA of good quality and
yield for use in molecular studies. The principle
modification in the described method includes use of
higher CTAB concentration, addition of PVP,
precipitation of DNA with sodium chloride and sodium
acetate and then binding of the initial crude CTAB
nucleic acid pellet to the silica matrix. These
modifications consistently produced pure and high
quality DNA suitable for further molecular analysis.
_______________
*Author for correspondence:
Telefax: +91-294-2410300
E-mail: [email protected]
Abbreviations: CTAB- Hexadecyltrimethyl ammonium bromide,
TE buffer- Tris EDTA buffer, EDTA- Ethylene diamine
tetraacetic acid, RAPD- Random amplified polymorphic DNA.
Materials and Methods
Plant Material
Plant material for DNA isolation was collected
from the identified wild populations of B. serrata.
Young leaves were taken from randomly selected
individuals. Leaves after collection were brought to
68
INDIAN J BIOTECHNOL, JANUARY 2012
laboratory in liquid nitrogen and stored at –20°C. The
leaves were subjected to five published genomic DNA
extraction protocols, which comprised of SaghaiMaroof et al1 (Protocol 1), Dellaporta et al2 (Protocol 2),
Bekesiova et al9 (Protocol 3), Storchová et al10
(Protocol 4), Unmodified Gilmore et al11 (Protocol 5)
and modified Gilmore et al11 (Protocol 6) developed
in our laboratory. The procedure for this protocol has
been described in this paper.
DNA Isolation Protocol
The young leaves (1 g) were deveined and grinded
to a fine powder in a mortar and pestle using liquid
nitrogen. The powder was transferred in 10 mL of
CTAB buffer containing 100 mM Tris (pH 8.0),
20 mM EDTA (pH 8.0), 1.4 M NaCl, 2.5% CTAB
(w/v), 1% PVP and 10 mM β-mercaptoethanol
(added freshly). It was mixed vigorously by vortexing
and incubated at 60°C for 30 min, followed by
treatment with equal volume of chloroform:isoamyl
alcohol (24:1). The upper phase, obtained by
centrifugation at 5125× g for 15 min at room
temperature, was transferred to a fresh autoclaved
centrifuge tube and then 1/10 volume of 3 M sodium
acetate (pH 5.2) and 1/2 volume of 5 M NaCl was
added to it. DNA was precipitated using 0.6 volume
chilled isopropanol and pelleted by centrifugation at
5125× g for 10 min at 4°C. The supernatant was
decanted and the DNA pellet was washed with 70%
ethanol. Crude CTAB DNA pellet was air dried and
suspended in 500 µL of 0.5 mL high-salt TE buffer
(10 mM Tris, pH 8.0, 1 mM EDTA, 1 M NaC1), with
brief heating at 65°C, if necessary. For some samples,
extra buffer was added to dissolve, as far as possible,
the large, carbohydrate-rich pellets. To each 1 mL of
crude nucleic acid extract, 3 volumes of binding
buffer [50 mM Tris (pH 7.5), 6 M NaC1O4, 1 mM
EDTA] was added and allowed to stand for 20 min.
The mixture was centrifuged at 550× g for 10 min and
the supernatant was transferred to a clean
polypropylene centrifuge tube. To this 300 µL of
diatomite suspension was added and tube contents
were mixed for 20-30 min by regular gentle inversion
to allow the binding of DNA to the diatomite. The
mixture was centrifuged at 550× g for 10 min and
supernatant was discarded. Then the diatomite was
gently re-suspended in 1.5 mL of wash buffer 1
(3 volume binding buffer, 1 volume water), followed
by centrifugation at 3000× g for 15 sec. The diatomite
pellet was again re-suspended in 1.5 mL of wash
buffer 2 [l volume 40 mM Tris (pH 8.0), 4 mM
EDTA, 0.8 M NaC1, 1 volume ethanol] and
centrifuged at 3000× g for 15 sec. The supernatant
was decanted and the DNA diatomite pellet was air
dried and suspended in 300 µL TE buffer (10 mM
Tris, pH 8.0, 1 mM EDTA), then tube was incubated
with regular inversion at 40-50°C for 20-30 min. The
tubes were centrifuged at 11600× g for 60 sec and
supernatant was collected into a clean 1.5 mL microfuge
tube. The supernatant was precipitated by addition of
1 M NaCl and 500 µL isopropanol and stored at −20°C
for at least 2 h, followed by centrifugation at 11600× g
for 5 min. The supernatant was discarded and the pellet
was air dried. The pellet was re-dissolved in 100 µL
0.1× TE buffer and stored at −20°C.
Preparation of Diatomite Suspensions
1. Diatomite (Hi-Media) was suspended in approx.
50 volume of water by lightly grinding with a pestle
in a glass mortar. The suspension was decanted,
avoiding any large grains or heavy sediment.
2. Diatomite was then allowed to settle down under
gravity for several hours and supernatant was
decanted with very fine material. The diatomite
was re-suspended in water and step was repeated
until superfine particles had been removed.
3. The diatomite was then collected by low speed
centrifugation and washed successively for 15 min
with continuous stirring in 0.1 M HCl and two
changes of water, centrifuging between each step.
4. It was re-suspended in water and autoclaved. Then
it was washed twice in sterile distilled water and
finally re-suspended in one volume of sterile,
de-ionized distilled water.
5. Aliquots were made and stored at 4°C. These were
vortexed before use to create a uniform suspension.
(Storage of a working aliquot in a conicalbottomed polypropylene centrifuge tube assists
rapid suspension)
Gel Electrophoresis
The quality of DNA was checked by agarose
(Hi-Media) gel electrophoresis. Samples were
prepared by taking 4.0 µL of DNA and 1.0 µL of
10× bromophenol blue dye (0.25% bromophenol blue
and 50% glycerol) on a ParafilmTM strip and mixed well
with the help of a micropipette. Samples were subjected
to electrophoresis in 1× TAE buffer for ~1 h at 80 V on
a 0.8% agarose gel matrix. Gels were photographed
under UV light using a Gel Documentation System
(DP 001 FDC, Consort, Belgium). All reactions were
repeated thrice to confirm the results.
SHARMA & PUROHIT: DNA ISOLATION FROM POLYSACCHARIDE RICH LEAVES
DNA Quantification
69
RAPD profiles were produced through PCR
amplification using the protocol described by
Williams et al12 with minor modifications. PCR
reactions were carried out in 0.2 mL polypropylene
PCR tubes (Bangalore Genei, India) using thermal
cycler (Master Cycler Personal, Eppendorf). Each
20 µL reaction mixture contained 1× Taq buffer
(100 mM Tris-Cl (pH 9.0), 500 mM KCl, 15 mM
MgCl2 and 0.1% gelatin), 2.5 mM MgCl2, 0.2 mM
dNTPs (Bangalore Genei, India), 20 pmol
oligonucleotide primers (Sigma Genosys, India),
1 U Taq DNA polymerase (Bangalore Genei, India)
and 30 ng template DNA. The reactions were
subjected to initial denaturation at 94°C for 4 min,
followed by 40 amplification cycles, each consisting
of 1 min at 94°C (denaturation step), 1 min at 37°C
(annealing step) and 2 min at 72°C (extension step)
with a final extension of 7 min at 72°C. The
amplification products were separated on 1.5% (w/v)
agarose gel, and stained with 0.5 µg/mL ethidium
bromide solution. DNA ladders of 1 Kb and 100 bp
(Bangalore Genei, India) were mixed and used as mol
wt marker for comparison of amplified products. Gels
were photographed as mentioned earlier. All reactions
were repeated thrice to confirm the results.
several protocols of genomic DNA extraction were
tested for yield, quality and suitability of DNA for
RAPD analysis in B. serrata.
The CTAB based protocol 1 yielded dirty yellow
DNA with high viscosity. In this protocol
polysaccharides co-precipitated with DNA on
addition of isopropanol and formed sticky brown
coloured pellet. The isolated DNA pellet could not be
dissolved in TE buffer and when subjected to
electrophoresis formed smear, indicating degradation
(Fig. 1A). In Protocol 2, CTAB was replaced by
anionic detergent SDS, but this also resulted in
recovery of DNA contaminated with polysaccharides
making it opaque and difficult to dissolve. Such DNA
samples stuck to the wells during electrophoretic
separation (Fig. 1B). To overcome this problem
various modified protocols were tried. These
modifications included use of detergent sodium
N-lauroyl sarcosine in extraction buffer (Protocol 3)
and grinding of leaf samples in sorbitol extraction
buffer (Protocol 4). However, these modifications
tried alone or in combination either yielded low
quantities of DNA or the DNA samples were of poor
quality unsuitable for electrophoresis, PCR
amplification and restriction digestion. Some
improvement in results was obtained from Protocol 5.
Although DNA isolation from this method resulted in
intact DNA without RNA contamination but gave
decreased DNA yield and quality (Table 1) and the
final DNA preparation was slight yellow in colour.
In marked contrast, the DNA purified by
Protocol 6, including CTAB based DNA isolation
with modifications and diatomite binding, yielded
Results and Discussion
The isolation of high quality genomic DNA,
essential for several molecular biology applications,
has always been not possible for many medicinally
important plants due to presence of secondary
compounds. However, standard CTAB methods of
Saghai-Maroof et al1 and Doyle & Doyle13 were
successfully applied for the extraction of DNA to a
number of plant species, such as, blackcurrant14,
conifers15 and ferns16, having secondary metabolites.
During the present investigation, none of these
described protocols were found appropriate for
extracting high quality DNA from the gum containing
leaves of B. serrata. The leaves contain high
polysaccharide contents, gums and resins, which
interfere in the amplification reactions, making the
DNA useless for research applications. Therefore,
Fig. 1—Gel images of total genomic DNA isolated from leaves of
B. serrata: A. Protocol 1, B. Protocol 2, & C. Restriction
endonuclease digestion pattern of genomic DNA isolated from
B. serrata using the Protocol 6 (M: Mol wt marker, phage DNA
Hind III digest, Bangalore Genei; Lane 1: Undigested DNA; &
Lane 2: EcoRI)
DNA
concentration
was
estimated
spectrophotometrically (UV-Vis Spectrophotometer,
Pharmaspec UV-1700, Shimadzu, Japan) by
measuring the absorbance at 260 nm. The original
DNA samples were then diluted to 10 ng/µL for PCR.
Conditions of PCR Amplification
INDIAN J BIOTECHNOL, JANUARY 2012
70
white, translucent DNA without any discolouration.
Quality of DNA was also confirmed through
spectrophotometeric analysis that showed absorbance
ratio between 1.75-1.85 when measured at two
different wavelengths (A260/A280), ensuring that the
DNA samples were free from protein and RNA
contamination and were PCR amplifiable. Therefore,
Protocol 6 was finally selected for all further
extractions of DNA. The average yield of DNA was
20-40 µg/g of fresh wt of tissue. Though the yield was
less than those obtained with other protocols but the
purity was much better (Table 1).
Samples
of
all
diatomite-purified
DNA
preparations from Protocol 6 when electrophoresed on
0.8% agarose/ethidium bromide gels (Fig. 2), most of
Table 1—DNA yield and purity obtained from B. serrata using
various protocols
No.
Protocol
Yield
(µg/g of
leaf
tissue)
OD
(260/280
ratio)
Reference
1
Protocol 1
65 -85
1.1-1.4
Saghai-Maroof
et al1
2
Protocol 2
18 -38
1.2-1.3
Dellaporta et al2
3
Protocol 3
1 -3
0.9-1.0
Bekesiova et al9
4
Protocol 4*
-
-
Storchová et al10
5
Protocol 5
3-9
1.5-1.6
6
Protocol 6
20-40
1.75-1.85
Unmodified
Gilmore et al11
Modified
Gilmore et al11
the samples showed an intensely fluorescing band of
about 50 kb. None of the samples showed evidence of
RNA contamination. Recovered DNA samples when
digested with the restriction endonuclease EcoRI,
showed complete digestion (Fig. 1C, Lane 2).
Genomic DNA samples when subjected to RAPD
analysis, decamer primers produced clear, scorable
and highly reproducible bands. Fig. 3 shows RAPD
profiles of B. serrata DNA, which indicates that the
DNA extracted by this method was suitable for PCR
amplification. The principle modification in this
method included use of higher CTAB concentration
(2.5%), addition of 1% PVP, precipitation of DNA
with 2.5 M NaCl and 0.3 M sodium acetate, and then
binding of the initial crude CTAB nucleic acid
complex pellet to the silica matrix in a solution of
chaotrope, followed by washing the silica
matrix/DNA with ethanol to remove the chaotrope
and finally the elution of DNA with only 300 µL of
TE buffer from the matrix. In this reaction, higher
CTAB concentration binds to polysaccharides and
addition of PVP prevents the browning of DNA
caused due to oxidation of polyphenols. Precipitation
of DNA with 2.5 M NaCl facilitated the removal of
polysaccharides by increasing their solubility in
isopropanol so that they do not co-precipitate with the
DNA. The addition of 0.3 M sodium acetate was
found useful in the formation of non-gelatinous
CTAB/DNA pellet and removal of colour
contaminants, resulting in the precipitation of white in
place of coloured pellet. The elution of DNA once
*The data is not available because of high viscosity
(unmanageable in pipetting) of sample so could not be
subjected to quantification.
Fig. 2—Gel image of total DNA isolated from leaves of B. serrata
using Protocol 6
Fig. 3—RAPD profiles of B. serrata generated from selected
primers (M: 1 kb ladder; & Lanes: 1. RUF 202, 2. RUF 203, 3.
RUF 205, 4. RUF 206, 5. RUF 209, 6. RUF 210, 7. RUF 211, 8.
RUF 214, 9. RUF 216, 10. RUF 217, 11. OP 1, 12. OP 7, 13. OP
8, 14. OP 12, 15. OP 13)
SHARMA & PUROHIT: DNA ISOLATION FROM POLYSACCHARIDE RICH LEAVES
only with 300 µL TE buffer significantly increased
the yield and quantity of DNA because again pooling
of the supernatant with more of TE buffer resulted in
too much binding of diatomite with DNA and
increased the probability that a single DNA molecule
will bind to two or more diatomite particles forming
bridges, which will break on centrifugation and
degrade the DNA more than necessary.
In conclusion, the only modification that proved
successful for DNA isolation from leaves of
B. serrata was CTAB based DNA extraction with
modifications and purification dependent on the
selective binding of DNA to a silica matrix of
diatomite in the presence of a chaotrope NaClO4. This
method could be adopted as standard method for
isolation of DNA from B. serrata or similar materials
rich in gum like polysaccharides. The diatomite
procedure described here is quick, simple and most
reliable enabling the processing of large number of
samples with ease.
Acknowledgement
Authors are thankful to Sir John A. Thomson,
Professor Emeritus, University of Sydney, and
Honorary Research Associate, National Herbarium of
New South Wales, Sydney, Australia, for his constant
guidance and comments. The Junior Research
Fellowship awarded to PS by Council of Scientific
and Industrial Research, New Delhi, is thankfully
acknowledged.
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