Scholarly Journal of Agricultural Science Vol. 2(6), pp. 242-248, June, 2013
Available online at http:// www.scholarly-journals.com/SJAS
ISSN 2276-7118 © 2013 Scholarly-Journals
Full Length Research Paper
Introducing a new method of genomic DNA extraction
in dicotyledonous plants
Hosseinpour N. Azad and G. H. Nematadeh*
Plant Genetic Engineering, Sari University of Agriculture and Natural Resources, Sari, Iran.
Accepted 2 June, 2013
Basically, for preparation of Deoxyribonucleic acid (DNA) extraction buffer, various materials were
used. Some of these materials are sensitive to temperature and others such as phenol- chloroform are
dangerous to human health. Moreover, they need liquid nitrogen and RNase enzymes; many of the
mentioned substances were removed using Sodium-Tris-EDTA (STE) buffer. In previous methods, we
needed substances such as BSA, also having high cost and sensivity to increase of temperature while
extraction process make it inevitable to use centrifuge device with cooler system. Most laboratories do
not have these facilities. Besides, common methods need to employ solutions in high volumes, which
all together increase the cost of DNA extraction. For this reason, using common methods to extract
DNA is not affordable in the laboratories with least facilities. In optimized and proposed method, we
used polyvinyl pyrrolidone (PVP) instead of Bovine Serum Albumin (BSA) in different experiments. In
addition to having high resistance in front of temperature increase, secondary metabolites are removed,
especially; polyphenols from cellular extracts of plant tissues also using this substance to reduces cell
extract washing time with component solutions of chloroform- isoamyl alcohol to one stage. The
aforementioned factors are the basis for the application of this method as a shorter method in
comparison with the common methods of DNA extraction. In addition, reducing the component to less
than a quarter of the previous methods, the STE method was optimized as a simple, fast and cheap
method to extract genomic DNA in a wide range of medicinal plants.
Key words: Deoxyribonucleic acid (DNA) extraction, medicinal plant, Sodium-Tris-EDTA (STE) buffer, polyvinyl
pyrrolidone (PVP).
INTRODUCTION
High contents of polyphenolics and polysaccharides in
plant leaves were problematic during the isolation of highquality/-quantity intact genomic nucleic acids (Michiels et
al., 2003; Karaca et al., 2005; Angeles et al., 2005;
Jabbarzadeh et al., 2009). Many different methods were
suggested for DNA extraction from various plant species
with high contents of secondary metabolites,
polyphenolics and polysaccharides. Some protocols
adopted the use of reducing agents such as dithiothreitol
and sodium metabisulfite while others successfully used
the common Cetylmethyl-ammonium bromide (CTAB)
associated with high salt as a reducing agent and
selective precipitant of nucleic acids and polysaccharides
(Sarwat et al., 2006; Križman et al., 2006; Dehestani and
*Corresponding author. E-mail: [email protected].
Tabar, 2007; Sharma et al., 2008; Dhakshanamoorthy
and Selvaraj, 2009; Hu et al., 2009).
Many studies for DNA extraction from ornamental and
medicinal plants were applied. Doyle and Doyll (1990)
method have been used to extract DNA from some
horticultural plants such as apple and grapes due to
special conditions in the tissues of medicinal plants which
has different levels of secondary metabolites, especially,
polyphenols extracting genomic DNA with appropriate
quality and quantity for Polymerase Chain Reactions
(PCR) faced with numerous problems. Perhaps many
students and researchers should spend more time for
optimization of DNA extraction procedures.
Although, several methods were used for the
extractions of genomic DNA in different medicinal plants
but till now, there is no report about optimization of
genomic DNA extraction method with STE buffers
(Hosseinpour et al., 2012). With regard to the high
Hosseinpour and Nematadeh
243
Table 1. Required materials for STE method.
S/No.
1
2
3
4
5
6
7
8
9
10
Materials
EDTA-Na2
Sucrose
Tris-HCL
SDS
LICL
PVP
Β-mercaptoethanol
Chloroform-isoamyl alcohol
Ethanol
Isopropanol
Used concentration
20 Mm and pH 8
4M
2.8 mM, pH
20%
8M
1/5%
0/2%
24:01
70%
Required amount
STE buffer: (EDTA 20 Mm and PH 8), Tris-HCL (2 M and PH 8) and Sucrose (4
M).
importance of medicinal plants, it is essential to extract
desirable DNA from these plants to analyze plant genetic
diversity and biological investigations. Avoidance of
freeze grinding tissues before addition of extraction buffer
was recommended, especially, for tissues with high
contents of water (Križman et al., 2006).
The purpose of this study is to make adaptive
comparison for Dellaporta, Cetylmethyl-ammonium
bromide (CTAB) and optimized Sodium-Tris-EDTA (STE)
method with each other and choose the best method in
extracting genomic DNA with appropriate quality and
quantity in some medical plants.
MATERIALS AND METHODS
The fresh leaves were transferred to the laboratory under
4°C. Then, plant materials were washed with distilled
water so as to avoid external contaminations. Plant
materials abandoned to evaporate water from these
surfaces were then used to extract DNA. Table 1 shows
the chemical materials and reagents used.
Extraction process
The extraction process is as follows:
- Make 500 mg of leaf tissue into small pieces and put
them into the mortar.
- Add 2 to 5 ml of STE buffer to the mortar and
immediately, add 300 µl of SDS (20%) to the mortar.
- Transfer 500 µl of the homogenized contents into the
vial with 1.5 ml volumes.
- Add 200 µl LiCl (8 M) and β- mercaptoethanol (0.2%)
into the all of the vials, (It is important to apply precaution
in using LiCl since anything more than advised content
inhibits DNA replication in PCR process).
- Add PVP (1.5%) to each vial (it should be done before
putting them in warm water as a powder and shake them
gently). Note: if plant materials have a high amount of
polyphenols, we can use more PVP.
- Take the vials in warm water (60°C) for 45 min (in every
10 min, it should be shaken gently).
- Remove vials from warm water and allow cooling at
room temperature.
- Add chloroform- isoamyl alcohol into the vials in equal
amounts with the content.
- Centrifuge the samples in 13400 rpm in room
temperature for 15 min.
- In the absence of solution transparency, add
chloroform- isoamyl alcohol with the same amount and
centrifuge the samples in 13400 rpm for 10 min.
- Supernatant the upper phase and add two third of the
isopropanol solution volume (-20°C) to each vials.
(Attention: After the eleven stages, DNA spindles will be
clearly visible in the middle of the solution, then, we can
continue the DNA extraction using the following two
methods):
First method
- Take DNA spindle with Pasteur pipette or any glass with
top capillary capability.
- Wash DNA with 70% alcohol (be careful about not
removing the pipette tip coils of DNA with alcohol).
- Place pipettes vertically to evaporate ethanol.
- Dissolve the obtained DNA in 100 µl of TE buffer,
(preheating TE buffer help influences DNA dissolving).
- DNA stored at 4°C until use.
Second method
- Centrifuge the samples for 10 min in 5000 rpm.
- Empty the fluid inside the tubes (Be careful so as not to
remove the pellets).
- Add 100 µl of ethanol 70% into each tube then,
centrifuge in 2400 rpm for 90 s (twice).
Scholarly J. Agric. Sci.
244
Table 2. Materials that used in three compared methods.
Methods
Materials
STE
+
+
+
+
+
+
Sucrose
Tris-HCL
SDS
LICL
NACL
CH3OOK
CH3OONa
Nitrogen
CTAB
RNase
EDTA
PVP
CTAB
+
+
+
+
+
+
+
+
Dellaporta
+
+
+
+
+
+
+
-
Table 3. Comparison of genomics DNA quality and quantity from young and matured
leaves using spectrophotometric method.
Method
STE
STE
Leaves samples 100 g
Young
Adult
*
*
CTAB
CTAB
*
Dellaporta
Dellaporta
*
DNA (µg/µl)
A260/A280
0.78 -0.91
0.30 -0.43
1.85
1.6
*
0.34 -0.41
0.18 -0.25
1.58
1.38
*
0.12 -0.19
0.03 -0.07
1.58
1.38
- Dry DNA at room temperature until the entire alcohol
content evaporates.
- Place pipettes vertically to evaporate ethanol and
dissolve the obtained DNA in 100 µl of TE buffer,
(preheating TE buffer help influences DNA dissolving).
Finally, autoclave TE buffer with PH 8 including (Tris 10
mM + EDTA 1mM).
DNA quantification
After extraction of genomic DNA with compared methods,
the obtained DNA were put in 100 µl of double distilled
water and kept in the laboratory at room temperature
overnight. The quality and quantity of extracted genomic
DNA was examined on 0.8% agarose gel and
spectrophotometer. Some of the samples in PCR process
levels do not show appropriate amplification. To make
sure that the desirable quantity and quality of DNA is
used, the template DNA was digested with EcoR1
restriction enzyme (Figure 3).
In addition, DNA purity was evaluated by spectrophoto-
-meter; DNA samples were measured after getting
dilution in 260 nm (the wavelength of nucleic acid)
absorption and their concentration was determined using
the following formula:
DNA (µg) = (A260) (dilution factor) 50.
Each absorption unit in 260 nm wavelength is equivalent
to 50 µg/µl of double- strand DNA, if the ratio of DNA
solution absorption (260/280) ranged from 1.7 to 1.9, the
quality of obtained DNA is desirable and it will be suitable
for PCR as a template DNA (10).
Through running of the DNA on each 0.8% agarose gel
wells, the band quality of each sample was specified. For
each sample, 4 µl of extracted DNA was mixed with 2 µl
of dye and was loaded in agarose gel wells at the
situation of TBE loading buffer. The running was taken
with constant voltage of 85 for an hour. After staining the
gel in Ethidium bromide, separated DNA bands were
observed and photographed under UV light by Gel
documentation system (UVP, USA, Co.). After the gel
image interpretation, the appropriate samples were
Hosseinpour and Nematadeh
245
Figure 1. DNA extracted from 100 g of young leaf from E. amoenum. (M) refers to 1 kb GeneRulerTM DNA ladder. Mix
(Fermentas, USA), S (STE), C (CTAB) and D (Dellaporta).
selected as template DNA for Polymerase Chain
Reaction (PCR).
RESULTS
The spectrophotomeric results obtained for each of the
two leaves samples (young and matured) showed less
amount of extracted DNA from matured leaves in each of
the three compared methods (Table 3). The physiologic
age of plant substances and DNA extraction methods are
effective in DNA quality and quantity. However, obtained
results showed that extracting genomic DNA with
optimized STE method for young and matured leaves
have better quality and quantity than other methods. In
0.5 g of leaf samples, the most amount of DNA was
obtained from optimized STE method with approximately,
780 to 910 ng/µl for young leaves and 300 ng/µl for
matured leaves, whereas, the least amount was related
to the Dellaporta method.
In CTAB procedure with the equal amount of leaf
tissue, the most amount of genomic DNA was
approximately obtained was 300 ng for young tissues and
200 ng for matured leaf tissues in per microliter of
extracted DNA. Some processes adopted the use of
reducing agents such as dithiothreitol and sodium meta
bisulfate while others successfully used the common
CTAB associated with high salt as a reducing agent and
selective precipitant of nucleic acids and polysaccharides
(Dhakshanamoorthy and Selvaraj, 2009; Hu et al., 2009).
In comparison with other studied methods using
Dellaporta method, the most amount of genomic DNA
was obtained in 150 ng for young leaves and 50 ng for
matured, respectively (Table 3). Jorge et al. (2005)
reported the yield of DNA ranged from 0.047 to 0.175
mg/g of fresh weight from Coconut samples.
The results obtained from quality measuring with
spectrophotometric showed the relative domination of
optimized STE method in access to genomic DNA with
the most amount, also, quality control test by agarose gel
of 0.8% confirmed this (Figures 1 and 2). In the analysis
of quality control with the information obtained from
spectophotometry, the average of absorption index was
located between 260 to 280 nm; results also showed that
Dellaporta method with 0.8 to 1.3 index is not an
appropriate method to extract genomic DNA from studied
plants, which was confirmed in electrophoresis (Figures
1and 2). The results related to enzymatic digestion tests
using EcoR1 enzyme showed that in optimized STE
method, DNA is digested appropriately and extracted
DNA with CTAB and Dellaporta procedures have taken
the next ranks, respectively. The suitable result from
Scholarly J. Agric. Sci.
246
Figure 2. DNA extracted from 100 g of mature leaf from E. amoenum. S (Optimized STE method), C
(CTAB method) and D (Dellaporta).
Figure 3. The enzymatic digestion test using EcoR1 restriction enzyme (from left to right), respectively,
optimized STE method and CTAB method; (Dellaporta) (the amount of injected DNA in each wells are
6 µl).
digestion process shows higher purity and better quality
of the DNA obtained from optimized STE method than
others (Figure 3).
In fact, the presence of impurities in the extracted DNA,
prohibited the restriction enzymes capability, thereby,
occupying probable restriction sites (Deshmukh et al.,
2007). The results of PCR showed that the DNA
extracted by optimized STE method has the necessary
Hosseinpour and Nematadeh
247
Figure 4. Young leaf genomic DNA extracted from some plants by STE method using RAPD primers (5- TCCGCTCTGG3); E (Echium amoenum), B (Borago officinalis), S (Senecio vulgaris), A (Aeluropus litolaris), and R (Oryza sativa).
quality for doing PCR operation. The resolution and the
number of amplified bands from genomic DNA extracted
from these plants in comparison with other methods
proved this (Figure 4).
DISCUSSION
The aforementioned set of factors caused extracted
genomic DNA from young leaves of studied plants to
have appropriate quality and quantity rather than other
parts of the plants. Young leaves have less
polysaccharides and secondary metabolites. This caused
the use of young leaves for DNA extraction in most cases
(Kumar et al., 2011). Finally, we can say that DNA
extraction from young and matured samples of plants
with desirable quality and quantity is counted as the best,
cheapest and fastest method for DNA extraction.
Chemical substances used for each of the three
studied methods are shown in Table 2. As seen in the
tables in optimized STE method, we do not need liquid
nitrogen to break cells and plant tissues, while on other
common methods, we need liquid nitrogen. Against
mentioned methods, we need sucrose. Using sucrose in
some extracting methods was also reported for the first
time. Kaufman et al. (1999) used sucrose buffer contents
in extracting genomic DNA from plant cells. It should be
mentioned that EDTA chelate ions of magnesium, cofactor for nuclease enzymes activity inhibit their activity.
Sodium Dodecyl Sulfate (SDS) is considered as a
detergent in the contents of DNA extraction buffers as a
remover of membrane lipids (breaking the cell and
nucleus wall), and also helps the separation of DNAprotein compounds (Sharma et al., 2008). Betamercapto
ethanol acts as anti oxidant and prevents oxidation of
polyphenol substances. Oxidation of polyphenolic
substances cause extracted DNA pellets to become
brown. Lithium Chloride (LiCl) was first used instead of
RNase by Prittila et al. (2001). Lithium constitutes a high
specific band with RNA which has over 200 bp and
makes Li- RNA complex such that this compound is
insoluble and can easily sediment, reducing the
contaminations of extracted genomic DNA. In addition,
using LiCl helps to remove restricted DNA (shared),
residual proteins, and polysaccharides. In plant species
which have high amount of polyphenolic substance, we
can apply an appropriate amount of PVP so as to
overcome the problems created by these substances in
the extraction process. PVP makes a complex hydrogen
band with polyphenols and simplifies their release from
DNA strands (Kadkhodaee, 2002).
Finally, we suggest optimized STE method as a simple,
fast and appropriate method for DNA extraction from
mentioned plants and some medicinal or crop plants with
fleshy tissues.
ACKNOWLEDGEMENTS
The authors would like to appreciate the honorable
Chairman of Tabarestan Institute of Genetic and
Agricultural Biotechnology for financial assistance offered
and all members of the genomics and Pharmacognosy
Laboratory staffs of Mazandaran University of Medicine
for their co-operation.
Scholarly J. Agric. Sci.
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