STAN DARD IZA TION OF PHE NOL FREE GENOM

HortFlora Research Spectrum

www.hortflorajournal.com

Vol. 6, Issue 3; 159-162 (September 2017)
NAAS Rating : 3.78

ISSN: 2250-2823

STANDARDIZATION OF PHENOL FREE GENOMIC DNA EXTRACTION OF POMEGRANATE GENOTYPES FOR DIVERSITY ANALYSIS
Dimpy Raina 1* and A. S. Sundouri 2
1

KVK Ferozepur, Punjab Agricultural University, Ludhiana-141 004, India
Faculty of Horticulture, Division of Fruit Science, SKUAST, Shalimar, Kashmir.
*Corresponding Author’s E-mail:
2

ABSTRACT : The isolation of intact high-molecular-mass genomic and good quality deoxyribonucleic acid
(DNA) is the pre-requisite for many molecular biology applications including long polymerase chain reaction
(PCR), endonuclease restriction digestion, southern blot analysis, and genomic library construction. The
presence of high concentrations of polysaccharides, polyphenols, proteins, and other secondary metabolites
in pomegranate leaves poses problem in getting good quality DNA. The study aimed to determine a reliable
and modified protocol based on the cetyltrimethylammonium bromide (CTAB) method for DNA extraction from
pomegranate leaves. Easy purification method was added to modify CTAB method using Tris-saturated
phenol: chloroform (1:1) and 3M sodium acetate. Polyvinylpyrrolidone (PVP) and β-mercaptoethanol were
employed to manage phenolic compounds. Extended chloroform-isoamyl alcohol treatment followed by
RNase treatment. Efficient yields of high-quality amplifiable DNA (200-1200 ng) was produced rapidly with
modified CTAB method. Quantity of obtained DNA from this extraction method was controlled in terms of
absorbance at wavelength of 260, 230 and 280 nm. The absorbance ratio of A260/A280 indicates presence of
dense protein. Spectrophotometric analysis at A260/A280 revealed ratio range of 1.77–1.94. The purified DNA

which has excellent spectral quality was efficiently amplified by 48 SSR primers and was suitable for
long-fragment PCR amplification.

Keywords : Polyphenolic compounds, genomic DNA, Tris-saturated phenol, chloroform, genetic diversity.
Pomegranate (Punica granatum L.), belongs to
the family Punicaceae, is one of the important and
oldest edible fruit, cultivated in Mediterranean
countries extensively in Iran, India, Pakistan,
Afghanistan, Saudi Arabia and in the sub-tropical areas
of South America (Elyatem and Kader, 3). In India, the
major pomegranate growing states are Maharashtra,
Karnataka, Gujarat, Andhra Pradesh and Rajasthan. In
recent years, pomegranate fruit is in demand
worldwide because of its superior pharmacological and
therapeutic properties. The arils and husk of
pomegranate fruit bear properties such as antioxidant,
anti-inflammatory, and anti-atherosclerotic against
some diseases (osteoarthritis, prostate cancer, heart
disease, HIV-1) (Malik et al., 7; Neurath et al., 10;
Sumner et al., 17). Due to its multipurpose medicinal
uses it is also known as “Super fruit” in the global
functional food industry (Martins et al., 8). The
germplasm of pomegranate needed to be evaluated
and conserved for its valuable properties. The
morphological characterization provided the inefficient
information about the germplasm so genomic based
Article’s History:
Received : 12-08-2017
Accepted : 02-09-2017

approaches have been taken to assess the intrinsic
knowledge about the germplasm. Genomic DNA
extraction is pre-requisite for the molecular
applications. The presence of high concentrations of
polysaccharides, polyphenols, proteins, and other
secondary metabolites in pomegranate leaves poses
problem in getting good quality DNA which is almost
insolvable in water or TE buffer, and inhibits enzyme
reactions (Reski, 13) and are also unstable for long
term storage (Sharma et al., 15).The present study
aimed to determine a reliable and modified protocol
based on the cetyltrimethylammonium bromide (CTAB)
method for DNA extraction from pomegranate leaves
with the addition of purification method Tris-saturated
phenol: chloroform (1 : 1) and 3M sodium acetate.

MATERIALS AND METHODS
Ten pomegranate genotypes viz. Kandhari, Moga
local, Assam local, Russian seedling, G-137, P-26,
Khug, Jhodpur white, Panipat selection, Kandhari
Kabuli were used as plant material for genomic DNA
extraction. The leaf samples of genotypes were
collected from germplasm maintain at New Orchard of
PAU, Ludhiana, India.


160

Raina and Sundouri

HortFlora Res. Spectrum, 6(3) : September 2017

Chemicals Used

Purification of Genomic DNA

An extraction buffer solution (pH 8.0) consisting of
1.5% CTAB (w/v), NaCl (1.4 M), Tris HCl pH 8.0 (100
mM) and EDTA pH 8.0 (20 mM), PVP (2 %),
â-mercaptoethanol 2 %. The other chemicals were
chloroform:Isoamylalcohol (24:1) v/v/v), iso-propanol,
Tris saturated phenol, phenol: chloroform: isoamyl
alcohol (25:24:1),Sodium acetate (3M) solution (pH
8.0),70% ethanol, RNase (10mg/ml of RNase in buffer,
10 mM Tris HCl, 15 mM NaCl pH 7.5) and TE buffer
(Tris HCl, 10 Mm pH 8.0, 1mM EDTA pH 8.0).

As the freshly isolated DNA contained certain
impurities like, polyphenolic, proteins, polypeptides etc.
it was necessary to purify the DNA before PCR
analysis. The DNA samples were thawed to room
temperature and an equal volume of Tris-saturated
phenol: chloroform (1:1) was added. The mixture was
then mixed thoroughly and centrifuged for about 5
minutes at 12000 rpm. The aqueous phase was pipette
out in a fresh tube and two chloroform: isoamyl alcohol
(24:1) extractions were performed as before. Both the
times, the mixture was centrifuged at 10,000 rpm for 10
minutes. The upper phase was again pipette out and
2.5 times the total volume of chilled ethanol were

added to it. The contents were mixed gently and the
precipitated DNA was spooled out. The extra salts were
removed by two washings with 70per cent ethanol at
10,000 rpm for 5 minutes and the pellet was dried at
room temperature. The pellet was dissolved in
appropriate volume of 1X TE. The DNA samples were
dissolved at room temperature and stored at -20° C
until used. Quantity of obtained DNA from this
extraction method was determined by spectrophotometer analysis at wavelength of 260, 230 and 280 nm.
The absorbance ratio of A260/A280 indicates presence
of dense protein. DNA concentration and purity was
also determined by running the samples on 0.8 %
agarose gel (Fig.1).

Isolation of Genomic DNA
Plant DNA was isolated using CTAB (Cetyl
Trimethyl Ammonium Bromide) (Saghai-Maroof et al.,
14) method with some modifications. Young leaves
from 10-12 years old trees were harvested, placed in
glassine bags and stored at –80°C until use. The
leaves were ground to fine powder using liquid nitrogen
to make leaves brittle as well as to stop DNase activity.
The powder was transferred immediately to a 50 ml
autoclaved Oakridge tube containing 20 ml of
pre-warmed (60°C) CTAB extraction buffer. The
powder was suspended in the buffer by inverting and
rotating the tubes gently. The tubes were incubated at
60°C for one hour in a water bath and were mixed
occasionally. After incubation, 15ml of chloroform:
isoamyl alcohol (24:1) was added and tubes were

swirled, till it made a dark green emulsion. The tubes
were placed on a rotary shaker for 30 minutes and then
centrifuged at 10,000 rpm for 10 minutes at room
temperature. Following centrifugation, the upper
aqueous phase was transferred to a clean sterile 50 ml
Falcon tube and 30 µl of sodium acetate (3M) was
added and contents were mixed thoroughly and
centrifuged at 12000 rpm for about 3 minutes. About 10
ml of chilled isopropyl alcohol was added to precipitate
the DNA and a good quality DNA floated at top was
hooked out using a sterile hooked Pasteur pipette. The
DNA was transferred into a clean sterile 2.0 ml
microfuge tubes rinsed with 70 per cent ethanol for five
minutes so as to remove any residual salts followed by
re-centrifugation. Pellet was collected and was allowed
to air dry (at room temperature) for one hour. Then
300-500µl volume of 1X TE was added and left for few
hours at room temperature. The dissolved DNA was
added with 10 µl of RNAse to each sample and
incubated at 370C in water bath for 45 minutes. The
samples containing DNA were stored at -20°C until
used.

PCR Analysis
A set of 25 SSR molecular markers ((Hasnaoui et
al., 5 and Pirseyedi et al., 11) was used for PCR
amplification. In vitro amplification using polymerase
chain reaction (PCR) was performed using 40 ng of
genomic DNA of each genotype in a final volume of
20µl per reaction containing 1 x PCR Buffer, 1.5mM

MgCl 2 , 200 µM dNTP mix, 1U Go Taq polymerase
(Promega), 0.5 µM of each single primer. Amplification
reactions were allowed to perform in a DNA
thermocycler (MJ Research) for 35 cycles after an
initial denaturation at 94°C for 4 minute. In each cycle
denaturation for 1 minute at 94°C, annealing for
1minute at (50-55°C) and elongation at 72°C for 1
minutes was performed with a final extension step at
72°C for 7 minutes. Negative control was used initially
to check the fidelity of the PCR reaction. The amplified
products were separated by electrophoresis on a 3%
agarose gel (0.03g/ml) ethidium bromide. Wells were
loaded with 20 µL of reaction volume. Electrophoresis
was conducted approximately 3hours at 90 volts, and
at the end, the gels were visualized and photographed
on an ultraviolet light transluminator.


Standardization of Phenol Free Genomic DNA Extraction of Pomegranate Genotypes for Diversity Analysis

RESULTS AND DISCUSSION
Molecular marker analysis in genome studies
greatly enhances the speed and efficiency of crop
improvement. The extraction of good quality and
quantity of DNA suitably meet the various molecular
based techniques for genome analysis. In the present
study DNA extraction was improved by modifying some
of the steps in the original CTAB DNA isolation protocol
given by Saghai-Maroof et al. (14). This procedure
resulted in extracting high quality, low-polysaccharide

genomic DNA from mature leaves of pomegranate.
Large quantities of polysaccharides are known to
interfere in many analytical applications and therefore,
lead to wrong interpretations (Singh et al, 16). CTAB
found to prevent the polysaccharide co-precipitations
(Dellaporta et al., 2).The presence of polyphenols can
reduce the yield and purity by binding covalently with
the extracted DNA, the mixing of PVP along with CTAB
might bind to the polyphenolic compounds by forming a
complex with hydrogen bonds and help in removal of
impurities to some extent. Similar residual phenols and
polysaccharides were removed and DNA was
precipitated selectively in the presence of high salts in
some woody plants (Fang et al., 4). Tannins, terpenes
and resins considered as secondary metabolites are
also difficult to separate from DNA (Mazid et al., 9).
Sodium acetate treatment fixed and removed tannins
and the extra secondary metabolites. The additional
step of purification with Tris Saturated Phenol :
Choloform (pH 8.0) followed by chloroform: isoamyl
(24:1) removed excess impurities of proteins,
polysaccharides and phenols. DNA isolation procedure
also yields large amounts of RNA, especially 18S and
25S rRNA (Joshi et al., 6). Large amounts of RNA in the
sample can chelate Mg 2+ and reduce the yield of the
PCR. A prolonged RNase treatment degraded RNA
into small ribonucleosides that would not contaminate
the DNA preparation and yielded RNA-free pure DNA.
Additional precipitation steps removed large amounts
of precipitates by centrifugation and modified speed

and time. Several plant DNA extraction protocols have
been reported in fruit crops like mango (Mangifera
indica L.) citrus (Citrus spp.), litchi (Litchi chinensis S.),
custard apple (Annona squasoma L.), guava (Pisidium
guajava L.) and banana (Musa spp.) (Porebski et al.,
12).The spectrophotometer analysis showed good
quality DNA (A 260 A 280 1.77–1.94) (Table 1). Cheng
et al. (1) also reported spectrophotometer analysis of
DNA extracted from old frosted citrus spp. leaves (A
260/A280 1.5 -1.87). The present modified protocol
was able to quantify high amount of DNA (200-1200
ng) on 0.8% agarose (Table 1 and Fig.1). DNA isolated

161

by this method yielded strong and reliable amplification
products showing its compatibility for SSR-PCR. For
SSR almost all the tested parameters like the primer,
Taq polymerase, dNTPs, magnesium chloride,
concentration of template DNA and temperature and
time intervals during denaturation , annealing and
elongation were optimized which also had an effect on
amplification, reproducibility and banding patterns (Fig.
2.). The optimized DNA isolation and SSR technique
may serve as an efficient tool for further genetic
studies.
Table 1 : Qualitative and quantitative differences in
genomic DNA of 10 pomegranate
genotypes extracted from mature leaves.
S.No.

Genotypes

DNA
concentration
(ng/2 µl DNA)

A
260/A280

1

Kandhari

300

1.92

2

Moga Local

200

1.94

3

Assam Local

300

1.89

4

Russian seedling

1200

1.77

5

G-137

1200

1.79

6

P-26

1200

1.77

7

Khug

1000

1.87

8

Jhodpur white

500

1.84

9

Panipat selection

1000

1.87

Kandhari Kabuli

1200

1.78

10

Fig. 1 : Electrophorotic separation of genomic DNA of
10 pomegranate genotypes. Numbers at the top of each
line are the genotypes as presented in Table 1.

Fig 2: In vitro amplification profile of 10 pomegranate
genotypes for SSR marker. Numbers at the top of each
line are the genotypes as presented in Table 1.


162

Raina and Sundouri

CONCLUSION
The results proved the reproducibility, reliability
and practicality of the modified protocol which yielded
high quality DNA on isolation and found accessible for
PCR analysis. It provides the opportunity to collect
good quality DNA from mature leaves from other
species high in polysaccharides and polyphenols.

Acknowledgements
Thanks
to
Inspire
Fellowship
Scheme
(Department of Science and Technology, Govt. of
India) for financial assistance and Molecular laboratory
of School of Agricultural Biotechnology, PAU for

technical advice.

9.

10.

11.

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q

Citation : Raina D. and Sundouri A.S. (2017). Standardization of phenol free genomic DNA extraction of
pomegranate genotypes for diversity analysis. HortFlora Res. Spectrum, 6(3) : 159-162.