Indian Journal of Biotechnology
Vol 9, January 2010, pp 69-73
Evaluation and optimization of DNA extraction method for Dalbergia sissoo leaf
H S Ginwal* and Shalini Singh Maurya
Division of Genetics and Tree Propagation, Forest Research Institute, Kaulagarh Road, Dehradun 248 195, India
Received 23 December 2008; revised 15 June 2009; accepted 28 August 2009
A modified protocol for Dalbergia sissoo genomic DNA isolation has been optimized based on a cetyl trimethyl
ammonium bromide (CTAB) method, described for other forest species. Leaves obtained from macro-propagated clones and
mature trees of D. sissoo were tested. The method involves mortar grinding of tissue, a modified CTAB extraction buffer
incorporating high salt concentrations, polyvinyl pyrrolidone and successive isoamyl alcohol-chloroform extractions with
modified temperature conditions. The modification involved the use of doubled concentration of polyvinyl pyrrolidone (4%
instead of 2%), increased incubation time with extraction buffer (40 min instead of 30 min), use of freshly prepared CTAB
buffer and increased timing of washing of DNA pellet with wash buffer (45 min instead of 30 min). The yield was
approximately 100 to 400 µg DNA per 100 mg of leaf tissue. The genomic DNA obtained by this method was suitable to be
used in RAPD and ISSR analysis. This extraction method would allow the molecular analysis of DNA from different clones
of D. sissoo.
Keywords: CTAB, Dalbergia sissoo, DNA extraction, forest tree, RAPD, ISSR
Introduction
Dalbergia sissoo Roxb., commonly known as
Shisham, is native to foothills of Himalayas of India,
Pakistan, Nepal and Indo-Gangetic basin. It grows in
the entire sub-Himalayan tract and also in the
Himalayan valley up to an elevation of about 1,500 m.
It naturally grows along streams gregariously on
alluvial soil and descends down in the plains to some
distance along stream banks. However, it is grown
throughout Indo-Gangetic plain and Rajasthan as
plantations. The species is used extensively for
timber, shelterbelts and fuel wood in the sub-humid
and drier areas. It’s a very important popular raw
material for wood based industries, especially
building construction and furniture because of
strength, pleasant brown colour and beautiful grains.
Being rich in protein, its leaves are suitable alternative
of cattle fodder during scarcity period. It also enriches
soil through atmospheric nitrogen fixation and rich
leaf fall.
Being an important timber species of the country,
lots of effort is being put for the genetic improvement
of D. sissoo. Knowledge of genetic relationship and
variation in the species is pre-requisite for the long
___________
*Author for correspondence:
Tel: 91-135-2755473; Fax: 91-135-2756 865
Mobile: 09412413158
E-mail: [email protected]; [email protected]
term breeding program, because it permits the
organization of germplasm including elite lines and
provides for more efficient parental selection1. A wide
array of techniques, including morphological and
biochemical, have been used in the study of
relationship and variation of forest trees, but the
assessment of phenotypic traits at times are time
consuming due to long gestation period of trees and
are not always reliable measure of genetic traits. The
analysis of DNA allows the direct assessment of
variation in the genotype and, hence, genetic analysis
of forest trees using molecular genetic markers
provides a powerful tool in selection of genetic
material in long rotation tree crops like Shisham.
DNA isolation and purification are important steps
for molecular biology studies. The quality and
integrity of the isolated DNA directly affects the
results of all subsequent scientific research. Like
reagents, good quality DNA is essential to achieve
good results in experiments2. Excess cell debris and
proteins may inhibit polymerase chain reaction
(PCR)3. Cellular components, polysaccharides,
polyphenols, etc., interfere with extraction and
purification of genomic DNA, which subsequently
influence DNA restriction, amplification and cloning.
For example, the presence of polysaccharides makes
genomic DNA highly viscous with glue like texture
that renders it unmanageable in pipetting and
70
INDIAN J BIOTECHNOL, JANUARY 2010
unsuitable for PCR by inhibiting Taq polymerase
activity4. On the other hand, polyphenols, in their
oxidized forms, covalently bind to DNA making it
useless for most research applications5,6. Avoidance of
extraneous cellular components has led to
development of several protocols for DNA extraction
from plants7-12.
Various protocols are employed because of
chemotypic heterogeneity among species. Even those
very closely related, a single DNA protocol may not
permit isolation of optimal genomic DNA13. The
situation is precarious for forest trees, which exhibit a
lot of variation within and between populations of a
single species14. For example, the quantity and purity
of genomic DNA in teak (Tectona grandis L.f.)
depends upon age and type of donor trees15. With
maturity, leaves contain increased quantities of
polyphenols and polysaccharides that prevent
extraction of good quality genomic DNA. To
overcome such problems, in the present study, authors
have standardized a procedure for the extraction of
good quality genomic DNA from young and mature
trees of D. sissoo for which no standard protocol was
present. The extracted DNA was further tested for
RAPD and ISSR analysis.
Materials and Methods
Leaves of mature trees (15-yr-old trees) and macropropagated clones (1-yr-old) of D. sissoo were
collected from the germplasm bank of Forest
Research Institute (FRI), Dehradun and stored at
–80°C before subjecting to for four CTAB based
protocols for genomic DNA extraction, which
comprised of Doyle and Doyle7 (Protocol 1), Stange
et al16 (Protocol 2), GeNei Spin Plant Genomic DNA
Prep Kit17 (Protocol 3) and Modified CTAB-polyvinyl
pyrrolidone (PVP)/ascorbic acid method (Protocol 4)
developed at our laboratory. Protocol 1 and 2
incorporated PVP-40 and β-mercaptoethanol but
Protocol 2 additionally had ascorbic acid and DIECA
in CTAB extraction buffer. Protocols 1 and 2 are
executed as described earlier7,16, while Protocol 3 was
executed following the instructions of the
manufacturer17. The procedure for Protocol 4 has been
described below.
Protocol 4
The young leaves (500 mg) without mid rib, veins
and margins were chopped and ground to fine powder
using liquid nitrogen in a pre-chilled mortar and
pestle. The fine powder was transferred to 2 mL
Eppendorf tubes containing 1 mL of preheated
(at 60°C) freshly prepared CTAB extraction buffer
(2% CTAB; 1.42 M NaCl; 20 mM EDTA; 100 mM
Tris HCl, pH 8.0; 4% w/v PVP40; 5 mM ascorbic
acid) and 3 µL of β-merceptoethanol. The mixture
was gently mixed by inversion to prepare slurry
and incubated for 40 min at 60°C in a water
bath. After incubation, equal amount of
chloroform:isoamylalcohol in ratio of 24:1 (v/v) was
added with gentle inversion for 15 min and
centrifuged for 5 min at 13,000 rpm at 25°C. The
upper aqueous layer was carefully collected and
transferred to another fresh Eppendorf tube. Again a
phase is separated with chloroform:isoamylalcohol
(24:1, v/v), as has been previously done, and the
upper phase was separated out. To this tube, 500 µL
of cold isopropanol was added. The mixture was
allowed to stand for 1-2 h at 4°C for precipitation of
fibrous nucleic acid, which was pelleted by
centrifugation for 15 min at 13,000 rpm. The DNA
pellet was washed with 998 µL of 76% ethanol and
2 µL of 5 M ammonium acetate, and incubated for
45 min at room temperature on gel rocker.
The mixture was again centrifuged for 5 min at
13,000 rpm, discarding supernatant. Again, the pellet
was washed with 70% ethanol, following
centrifugation for 15 min at 13,000 rpm. The
supernatant was discarded and the pellet was dried in
thermo-block. The dry genomic DNA pellet was
dissolved in 100 µL of Tris-EDTA buffer (10 mM
Tris-HCl; 1 mM EDTA; pH 8.0) and stored at –20°C.
The quantity (µg) of extracted genomic DNA was
estimated at 260 nm on Biophotometer (Eppendorf).
The purity of the DNA was assessed on agarose gel
electrophoresis (Biorad) (Fig. 1) as well as through
absorbance ratio 260/280 nm on Biophotometer
(Eppendorf). The deviation above and below from
value 1.80 at 260/280nm absorbance ratio determines
RNA and protein contamination in the extracted
genomic DNA, respectively18.
RAPD and ISSR Analyses
The extracted genomic DNA was tested for PCR
amplification using RAPD decamer arbitrary primer
(OPA 20 - GTT GCG ATC C) from a company
(QIAGEN Operon, 1000 Atlantic Avenue, Almeda,
CA 94501, USA) and ISSR primer (UBC 851 - GTG
TGT GTG TGT GTG TYG) from University of
British Columbia, Canada. The reaction mixture
(25 µL volume) for RAPD and ISSR assays consists
GINWAL & MAURYA: DNA EXTRACTION METHOD FOR D. SISSOO LEAF
of genomic DNA, 10× Taq buffer, 2.5 mM each of
dNTPs, 25 mM MgCl2, and 5 U of Taq DNA
polymerase. However, 4 ng genomic DNA was used
for RAPD assay and 25 ng for ISSR assay. The
ultra pure distilled water was used to make the
reaction volume.
For RAPD assay the first cycle was programmed as
denaturation for 1 min at 94°C, annealing for 1 min at
37°C and extension for 1 min at 72°C. Subsequently,
41 cycles each of denaturation was given for 45 sec at
94°C, annealing for 1 min at 37°C and primer
extension for 1 min at 72°C. An additional extension
at 72°C for 10 min was performed to facilitate
complete extension. For ISSR assay, PCR
amplification was carried out at 94°C for 5 min for
initial denaturation, followed by 35 cycles of
denaturation at 94°C for 30 sec, primer annealing for
30 sec at 54°C, extension at 72°C for 60 sec, and
termination by 5 min at 72°C.
The amplification products were electrophoresed
using 1.5% agarose gels with 1× TBE buffer at pH 8.0
for 3 h. Gel was visualized by 0.5 µg/mL ethidium
Fig. 1 (a-d)—Gel images of total genomic DNA isolated from
leaves of adult (lane 1) and juvenile (lane 2) D. sissoo trees:
Genomic DNA isolated following Protocol 17 (a), Protocol 216 (b),
Protocol 317 (c), and modified Protocol 4 (d).
bromide staining and photographed under UV light
using Gel Documentation System (GelDoc-It System,
UVP Ltd., Unit 1, Trinity Hall Estate, Nuffield Rd.
Cambridge, CB4 1 TG, UK). Reagents and chemicals
used in the present study were of analytical grades
and obtained from Sigma (Sigma-Aldrich, Inc., 3050
Spruce Street, St. Louis, MO, USA).
Results and Discussion
D. sissoo, being source of natural products mainly
wood and bioactive substances like isoflavonoids,
produces large amounts of secondary metabolites and
substances of medicinal or industrial importance.
Hence, DNA extraction from leaves of D. sissoo is
complicated by the abundance of secondary
metabolites. Also, the leaves exhibit oxidation when
left under humid conditions after collection, because
of which it is necessary to dry or freeze them as fast
as possible to preserve the quality of DNA19.
Age of tissue is important in determining DNA
yield and quality20. Leaves from juvenile D. sissoo
invariably extracted better yield and quality of
genomic DNA than those from mature D. sissoo. The
gel image of DNA extracted from mature tree leaves
showed a smear from high to low mol wt position
without having any intact band, indicating an
impression of degraded nature of DNA (Fig. 1). On
the other hand, Protocol 4 registered better values for
both parameters of genomic DNA than other three
protocols included in the study (Table 1). Genomic
DNA of both types of samples obtained in Protocol 4
also exhibited single consolidated band without
contamination of RNA (Fig. 1d) in comparison to the
remaining protocols (Fig. 1a-c) on agarose gel. DNA
yield and quality would likely to vary throughout the
year21, however as per our observation, the best
results were achieved when leaves were harvested in
spring season, i.e., March and April. At this time the
new flush of juvenile leaves appears which are
more suitable for obtaining good quality and quantity
Table 1—DNA extraction protocols and yield of extracted DNA from D. sissoo leaves
No.
1
2
3
4
Protocol
Protocol 17
Protocol 216
Protocol 317
Protocol 4
(modified)
From mature tissue
Yield
OD 260/280
(µg per 100 mg
ratio
of leaf tissue)
40 to 50
20 to 30
60 to 90
100 to 400
71
1.29 to 2.03
1.42 to 2.09
1.10 to 1.34
1.69 to 1.99
From juvenile tissue
Yield
OD 260/280
(µg per 100 mg
ratio
of leaf tissue)
50 to 60
20 to 40
50 to 200
100 to 400
1.58 to 1.97
1.64 to 2.01
1.65 to 1.97
1.71 to 1.90
72
INDIAN J BIOTECHNOL, JANUARY 2010
Fig. 2—Gel photograph of the genomic DNA of studied D. sissoo
clones isolated by optimized protocol
Fig. 3 (a & b)—a. RAPD pattern obtained from DNA extracted
from modified Protocol 4: фX-174 DNA/BsuRI (HaeIII)
molecular weight marker (lane 1); Eighteen DNA samples (lane
2-19) amplified using primer OPA-20. b. ISSR pattern obtained
from DNA extracted from modified Protocol 4: фX-174
DNA/BsuRI (HaeIII) molecular weight marker (lane 1); Eighteen
DNA samples (lane 2-19) were amplified using UBC primer 851.
of DNA. Sytsma et al22 has also emphasized the
use of fresh and young leaf tissue to obtain good
quality of DNA.
Out of the various protocols tried, the CTAB
method7,16 with modifications (Protocol 4) was found
suitable for extraction of desired quantity and quality
of genomic DNA from the leaf tissues. Approximate
100-400 µg of DNA/100 mg of foliage material was
obtained with this protocol from both juvenile
and mature tissues, which is enough to carry out
1000-1500 typical RAPD reactions. The principle
modification in this method included use of 4% PVP
instead of 2% PVP used in Protocols 1 and 2,
increased incubation time with extraction buffer
(40 min instead of 30 min), use of freshly prepared
CTAB buffer and an extended washing time (45 min
instead of 30 min) with wash buffer and a repeated
phase separation step with chloroform-isoamyl
alcohol. High PVP concentration removes the
polyphenols as it forms a complex with polyphenols
through hydrogen bonding, allowing them to be
separated from the DNA, and reducing levels of
polyphenol in the product23. Extended incubation and
washing durations coupled with the separation phase
appears to have excluded DNA contaminants like
polysaccharides, polyphenols and other secondary
metabolite lysates, which otherwise hamper DNA
isolation and purification.
The isolated DNA using Protocol 4 of various
samples of D. sissoo (Fig. 2) was tested in PCR
amplification for RAPD and ISSR profiling with
several primers. The PCR products produced by
RAPD and ISSR analysis shows clear banding
patterns (Fig. 3). Thus, this protocol proved to be
advantageous because of its simplicity and affordable
reagents, besides achieving intact high mol wt and
purity of genomic DNA. The isolated DNA proved
amenable to PCR amplifications including RAPD and
ISSR analysis. The DNA extraction protocol
described here consistently produced high yields of
pure genomic DNA, while allowing for high sample
throughput.
Acknowledgement
Authors are grateful to Punjab Forest Department,
Government of Punjab, India for providing necessary
financial support for conducting the present study.
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