This research note has been accepted for publication by Harmony Newsletter published online from Patna, Bihar, INDIA
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Extraction
of Genomic DNA from Lantana camara
Leaf: Simple Protocol for Freshers in Molecular Biology and Biotechnology
Sourav
Datta1, Suman Roy2, Sourav Kumar Das3, Palash
Mukherjee4 and SKT Nasar*
Department of
Biotechnology, Bengal College of Engineering and Technology
(Affiliated to
West Bengal University of Technology), Durgapur-713212, West Bengal
Abstract
We
present here an ultrasimple, effective and inexpensive procedure to extract genomic
DNA from plants and other organisms with special focus on a common weed Lantana camara L. This report also presents a brief discussion on some
selected downstream applications of genomic DNA extracted by this
protocol.
Introduction
Genomic
DNA comprising nuclear, mitochondrial and chloroplast DNA is basic to molecular
biology and biotechnology and application of genetic technologies. Extraction
of DNA from organisms is the first step for the application of molecular
biology. Shirazu et al. (2009), Nasar and Nasar (2009, 2010) and others demonstrated
that extraction of DNA from different organisms is easy, inexpensive and fun.
Lantana camara (L.) is a common
invasive weed plant showing wide diversity. Lantana
biodiversity has been used to produce commercially available ornamental varieties
for hedges and gardens. We have selected Lantana
camara for the present work for two main reasons: Durgapur shows wide
variability and DNA extraction is rendered difficult due to high content of pentacyclic
terpenoids and volatile oil in leaves.
We
show here that gDNA extraction from Lantana
leaf is effectively as easy as for other plant species. We also show that
its applicability in selected downstream procedures is feasible.
Materials and
Methods
Planting selected materials in
pots
Selected
disease-free plants of Lantana camera L.
were maintained live in earthen pots at the departmental garden. Healthy fresh
leaves were collected for DNA extraction experiments.
Extraction of genomic DNA
The
procedure standardised by Nasar & Nasar (2010) and Nasar, Mukherjee &
Trivedi (2010, unpublished; personal comm.) has been used here. This protocol
requires household items such as refrigerator, mortar-pestle, table salt, dish
washing liquid soap and alcohol.
The
method of gDNA extraction from leaf of Lantana
camera L. constituted the following steps:
(i)
All items – leaves, glassware, and chemicals – were pre-cooled in the freezing
chamber of a refrigerator and all steps were carried out on a bed of ice cubes
or in ice box; (ii) Only healthy and young leaves were collected and
washed with clean water (Fig 1 A); (iii) leaves were cut into small pieces and surface
dirt of the material removed by washing repeatedly with distilled or drinkable water (Fig
1 B); (iv) A tablespoonful of clean water, a pinch each of ethylenediaminetetraacetic
acid (EDTA) and Tata® salt (NaCl) were added to the material and it was pulverised
with mortar-pestle to make a paste of tissue; (v) The paste was filtered through
fibre-free cloth and the filtrate was collected in a test tube or small glass
container; the residue was discarded; (vi) 2-4 drops of Vim® liquid dish
washing soap were carefully added to the suspension of pulverised tissue; very
gentle stirring with a pre-cooled glass rod avoided lathering; (vii) Finally, pre-cooled
ethanol was poured slowly along the wall of test tube/container without disturbing
the tissue suspension; (viii) The test tube was kept in the refrigerator at
14-15O C for 20-30 minutes and progress was watched intermittently; (ix)
Bubbles began rising through alcohol layer raising DNA threads that aggregated
as a white cloud floating in the ethanol layer (Fig 1 C & D); (x) DNA, at
his stage, was carefully sucked out with a long-nozzle glass dropper and was then
expelled from the dropper in pre-cooled 70% ethanol in a glass container for
storing in a refrigerator (Fig 1 E).
Fig. 1. Steps of gDNA extraction
from leaves of Lantana camara L. from
preparing the material to DNA isolation to storing
Sufficient amounts of DNA were
collected in about 30 minutes as can be seen in Fig. 1 D and E.
Confirmation of
the extracted substance being DNA
Several protocols exist for
confirmation of the extracted substance being DNA. We used ethidium bromide
[EtBr] in a simple experiment. The extracted DNA stored in 70% ethanol was
poured on a clean watch glass (#1) and kept in a refrigerator for four-five
days till the alcohol evaporated. Air-dried extracted DNA clung to glass
surface at the centre. A drop of EtBr was placed over the substance. Another
drop of EtBr was placed on a clean watch glass (#2) to act as experimental
control. Watch glasses #1 and #2 were exposed to UV light in a transilluminator.
The fluorescence patterns were photographed [Fig. 2 (a)]. A teaspoonful of
water each was added to another set of #1 and #2 and similarly fluoresced [Fig.
2 (b)].
Fig. 2. Ethidium
bromide (EtBr) fluorescence and EtBr-stained air-dried DNA and DNA dissolved in
water
It was observed that EtBr alone
fluoresced with lesser intensity than in conjunction with the extracted
substance [Fig. 2(a) #2 and (b) #2]. EtBr is known to intercalate with dsDNA
and produce intense fluorescence. On the other hand, ssDNA, RNA and stretches
of dsRNA fluoresce with much lower intensity. This mini-experiment confirmed that the
extracted substance is indeed DNA.
Confirmation
of gDNA
Leaf cell contains nuclear,
chloroplast and mitochondrial DNA. It was important to verify if the extracted
DNA was a mixture of nuclear and organellar DNA since downstream applications
would depend upon this information. To
this end, the extracted DNA was subjected to agarose gel electrophoresis in the
laboratory.
Agarose gel
electrophoresis for analysis of gDNA
The basic information utilised
for this section was based on Ogden and Adams (1987) and Brody and Kern (2004).
The extracted DNA was placed in pre-cooled water in eppedorf tube and rinsed by
centrifugation several times before use. Washing with water would remove salt,
ethanol and lighter segments of sheared DNA,
The extracted DNA samples were
electrophoresed in 1% agarose gel with EtBr at 75 v and, 60 mA for 1 hr. The EtBr-DNA fluorescent bands under
transilluminator were captured in digital pictures.
Fig. 3. DNA bands of Lantana camara (L.) after agarose gel electrophoresis. Heavy band likely
nuclear DNA, Light band likely chloroplast band and Lightest band likely
mitochondrial DNA
The electrophoresis yielded three
distinct bands. A comparison of the results (Fig. 3) with other results showed that
Lantana leaf DNA bands were similar to those of DNA extracted from different plant
species. The heaviest band represents nuclear DNA (nDNA), the light band is
chloroplast DNA (ctDNA) and the lightest band comprises mitochondrial DNA
(mtDNA).
The result shows that extraction
of DNA from Lantana camara (L.) leaf
is easy, quick and inexpensive. It also confirms that the extracted ‘white
cloud’ in ethanol is indeed DNA as further confirmed by the electrophoresis result.
Discussion
Experiments
conducted at Department of Biotechnology, Bengal College of Engineering and
Technology, Durgapur, confirmed that the ultrasimple procedure of gDNA extraction from Aloe vera leaf (Mala 2010), Oryza
sativa leaf (Ganguly 2010; Tewari 2011), giant tiger prawn (Penaeus monodon Fab.) muscle tissue (Nasar and Trivedi, pers.
comm.), goat liver and Lactobacillus (Nasar and Mukherjee,
pers. comm.) are uniformly effective.
Four
basic steps are essential for all DNA extraction protocols: (a) breaking cell
membranes by grinding to expose cellular contents and DNA, (b) getting rid of
intracellular boundaries by disrupting lipo-protein membranes by a detergent
containing sodium dodecyl sulphate, (c) removing proteins by protease such as
papaya juice and (d) precipitating DNA with alcohol, preferably ethanol. The
experiment must be conducted in cool conditions to decrease endonuclease
activity. Procedures must be carried out gently to avoid shearing of DNA.
An
inexpensive gDNA extraction protocol (Shirazu et al. 2009; Nasar & Nasar
2010) that consumes less time without compromising with quality has been
considered handy for this work. This report has shown that the ultrasimple protocol
of gDNA extraction is inexpensive, quick and effective as compared with standard
procedures such as the hexadecyltrimethylammonium bromide (CTAB) method (Ausubel
et al. 1994, Tewari 2011) are elaborate, costly and time consuming. This report
corroborates our other experiments. The total time duration for the experiment
from harvesting plant tissues to harvesting gDNA has been found to be typically
less than an hour.
Extraction
of DNA from plant tissue varies with experimental materials. Required
modifications adopted for DNA extraction from Lantana camara without changing the fundamentals of different steps
yields sufficient amounts of gDNA amenable to downstream analysis.
Addition
of table salt in lieu of lab-grade NaCl is important. The negative charge of
one of the oxygen atoms linked to the phosphorous provides high polarity to DNA
that dissolves in water at neutral pH. The
negative charge of phosphodiester group of DNA is neutralised on addition of
salt. DNA then becomes much less soluble in water. Iodised Tata® table salt
contains enough NaCl for the purpose of the protocol under report. Table salt
worked well in all our experiments.
Ethylenediaminetetraacetic
acid (EDTA) provided better results when used in our experiments. EDTA is a
chelating agent for metal ions such as Ca2+, Mg2+ and Fe3+.
Nucleases need divalent cations such as Mg2+ which when depleted
deactivates the enzymes.
Experiments
reported here used Vim® dishwashing liquid soap containing sodium dodecyl/lauryl
sulphate (SDS/SLS). SDS in the soap is a strong anionic detergent that
solubilises and breaks lipo-protein membranes and nuclear envelope. In addition to breaking down membranes, SDS emulsifies lipid
bi-layer structure of cell and nuclear membranes. SDS also helps the
release of chromosomal DNA from histones and other DNA binding proteins by
denaturing them.
After
careful examination of DNA precipitation in isopropyl alcohol, denatured
alcohol or ethanol in pilot experiments, cold ethanol was found to yield the
best results. Addition of alcohol and
salt causes DNA to precipitate while other soluble cell components remain in
solution in the aqueous phase. Alcohol also removes alcohol-soluble-salt (see
Kurabo PI-80X 2010; CTAB, CIMMYT 2005).
Experiments
in our laboratory revealed three bands of DNA extracted from leaf of Rice (Oryza sativa L. cv. MTU 7029
‘Swarna’; Ganguly 2010 and Tewari 2011), Aloe
vera L. (Mala 2010) and Lantana camara L. (present report) as can be seen in the collage
presented at Fig. 4.
Ganguly
(2010), in another experiment on the DNA of fresh and senesced leaf and fresh
root of rice (Oryza sativa L. cv. MTU 7029) successfully showed that
the three bands as seen in Fig. 4 represent nuclear DNA (nDNA; heavy band),
chloroplast DNA (ctDNA; light band) and mitochondrial DNA (mtDNA; lightest
band). The parallel illustrated in Fig. 4 confirms that the ultrasimple simple
protocol is undeniably effective for gDNA extraction from Lantana leaf.
The
gDNA extracted by the ultrasimple procedure is fit for downstream application.
Tewari (2011) subjected gDNA extracted by standard (CTAB) and ultrasimple
protocols to digestion with two restriction enzymes i.e. Hind 111 and EcoR1 for
RFLP studies on rice (Oryza sativa L. cv. MTU 7029). He observed that gDNA
extracted by these procedures yielded the same results. Similarly, our PCR studies on gDNA extracted
by standard and ultrasimple protocols from giant tiger prawn (Penaeus monodon Fab.) muscle tissue (Nasar and Trivedi, pers.
comm.) showed equally acceptable result.
Fig.
4 (A, B, C & D) Collage of leaf DNA from (A & B) Oryza sativa L. (C) Lantana
camara L. and (D) Aloe vera L.
after agarose gel electrophoresis showing three bands each. (A & B) DNA bands
of Oryza sativa L. cv. MTU 7029 leaf; (A) DNA extracted by
the ultrasimple protocol as in the present report; (B) DNA extracted by standard
(CTAB) protocol; (C) DNA bands of Lantana
camara L.; Present work, DNA extracted by ultrasimple protocol under this
report; (D) DNA bands of Aloe vera L.;
DNA extracted by the ultrasimple protocol as in the present report;
(1%
Agarose gel, 20µl EtBr; electrophoresis running time 3 hr 30 minutes at 50
volts)
Although
gDNA extracted by ultrasimple protocol has an OD of 1.4-1.5 at 260nm/280nm UV absorbance
(Tewari 2011), our conclusion that the ultrasimple protocol for extraction of
gDNA good for downstream applications is inescapable. This is corroborated by
Chum et al. (2012) who showed that DNA extracted directly from tissues can be
used for PCR analysis.
A
major criticism of teaching in molecular biology and biotechnology is that
practicals are not conducted appropriately (Lakhotia 2008). Such inadequacies
are due to the prohibitive costs of infrastructure coupled with fund crunch.
Experiments in selected areas with innovative and cost cutting measures (Nasar
2009) can lessen the burden. The present work has been undertaken to address
this issue.
The
present work shows that, with a good understanding of fundamental principles of
protocols, innovative experiments in molecular biology and biotechnology can be
cost-effective and quick. Students can, for example, extract DNA at residences
and bring samples to laboratory for further analysis.
Acknowledgement
Authors
are indebted to Bengal College of Engineering and Biotechnology, Durgapur for
providing facilities and to Mr. Prasenjit Tewari, Mrs. Kanchan Mala and Mr.
Joydev Ganguly, all former students of SKTN, for allowing us to use data from
their dissertations.
References
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1, 2 & 3Former B. Tech.
(Biotechnology) students; 4Lab In-charge; 1sourav.aka.peter@gmail.com,
2suman.biotech1411@gmail.com, 3srvhell@gmail.com, 4palash_muk@rediffmail.com,
*skt.nasar@gmail.com;
Corresponding author, Former Honorary Professor (Biotechnology)
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