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Biotechnology DOI 10.1002/biot.200600195 Biotechnol. J. 2007, 2, 386392

Journal

Review

Plant genomic DNA isolation: An art or a science

Astha Varma1 , Harish Padh2 and Neeta Shrivastava1

1 B. V. Patel Pharmaceutical Education and Research Development (PERD) Centre Department of Pharmacognosy and
Phytochemistry, Ahmedabad, Gujarat, India
2 B. V. Patel Pharmaceutical Education and Research Development (PERD) Centre Department of Biotechnology, Ahmedabad,

Gujarat, India

Isolating quality DNA from tissues/cells presents a variety of problems in particular when plants Received 27 September 2006
are used as the source material. The specific characteristics of plants like the presence of rigid poly- Revised 11 December 2006
saccharide cell wall, pigments, chemical heterogeneity of secondary metabolites found in diverse Accepted 20 December 2006
species of plants, etc., necessitate special consideration and skill during isolation procedure. Un-
til now, numerous protocols have been published for the purpose, but none is found to be uni-
versally applicable. Various factors starting from the selection of source material to the concen-
tration of metabolites present in the plant decide the course of the isolation procedure. The pres-
ent review is an update of various methods used for plant genomic DNA isolation, and it epito-
mizes the various problems faced and the solutions made to contend with them during DNA
isolation from plant cells.

Keywords: Chemical heterogeneity Genomic DNA Polyphenolics Polysaccharides

Secondary metabolites

1 Introduction plant molecular biologists are facing daily, is the difficul-

The area of plant molecular biology has witnessed con-
tinuous advancements dealing with the study and ma-
nipulations of plant genetic resources at the molecular
level. New frontiers are being opened with each new dis-
covery, the latest significant achievement being the
completion of rice genome sequencing. The genetic ma-
terial has been manipulated to the core by researchers,
including production of transgenics and biopharmaceu-
ticals, molecular breeding, genome mapping etc. How-
ever, the problem with the recalcitrant targets, which
Correspondence: Dr. Neeta Shrivastava, B. V. Patel Pharmaceutical Educa-
tion and Research Development (PERD) Centre, Sarkhej Gandhinagar
Highway, Thaltej, Ahmedabad 380054, Gujarat, India
E-mail: neetashrivastava_perd@yahoo.co.in
Fax: + 91-79-27450449
Abbreviations: CTAB, cetyltrimethylammonium bromide; DIECA, di-
ethyldithiocarbamic acid; PVP, polyvinyl pyrollidone; PVPP, polyvinyl
polypyrollidone
ty in obtaining contamination free, high-quality genom-
ic DNA, without which any further analysis or manipula-
tions are not feasible. The purer the DNA, the more reli-
able the result becomes because contaminated nucleic
acid often fails to give precise, reliable and reproducible
results.
In the preceding two to three decades, researchers
have developed numerous protocols and procedures to
isolate quality DNA from a variety of plant species, which
can be used depending on the purpose of isolation. Some
widely followed procedures are those given by Murray
and Thompson [1], Doyle and Doyle [2], Rogers and Ben-
dich [3], Lodhi et al. [4]. These methods have been proven
successful in a number of experiments until now. Howev-
er, none of these methods, or any other published method
has established itself as universally applicable to all plant
varieties. In general, researchers need to modify a proto-
col or blend two or more different procedures to obtain
DNA of desired quality for a particular requirement. In this
review we have emphasized the factors that lead to such
modifications, and that are imperative during genomic
DNA isolation from plants.

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2 Major factors influencing isolation
methodology
Fundamentally, a genomic DNA isolation procedure from
any prokaryotic or eukaryotic cell includes three central
steps, (1) removal of envelops (cell walls/membranes)
around the DNA, (2) separation of DNA from all other cell
components (cell wall debris and metabolic substances),
and (3) maintenance of integrity of DNA during the pro-
cedure, i.e., protection from nucleases and mechanical
shearing [5]. There are numerous factors that influence
the isolation methodology, rendering the genomic DNA
isolation from plants, an art rather than a science.
2.1 The source material and processing
Presence of a rigid polysaccharide cell wall makes plant
cells relatively difficult resource to extract DNA. Thus, be-
fore deciding the course of extraction process from the
source plant material, some major factors need to be con-
sidered.
2.1.1 Tissue type and age
The age and the kind of the source tissue greatly affect the
quality and yield of isolated DNA. Young, healthy and ten-
der tissues, especially partially expanded leaves make an
ideal choice as they can yield good quality and quantity
of DNA due to a larger number of cells and less deposition
of starch and secondary metabolites [1, 2, 6, 7]. Etiolated
young leaves are also reported to be a good source mate-
rial for DNA isolation. Growing the source plant for 34
days in dark before harvesting the leaf tissue for isolation
makes the isolation procedure easier, as it etiolates the
leaves ([8, 9] and www.qiagen.com). In contrast, DNA ex-
tracted from mature leaves is reported to be of poor qual-
ity and low yield due to the presence of high concentra-
tions of polyphenols, tannins, polysaccharides and other
secondary metabolites, which either lead to embedding of
DNA into a sticky gelatinous matrix or form undesirable
brown colored products unsuitable for any application
[1013]. Apart from the leaves, other plant parts like stem,
roots, seeds, embryos, rhizomes, etc., can also be used de-
pending upon the source plant and availability of tissues.
2.1.2 Collection and storage
Proper handling and storage of the plant tissue after har-
vesting is an essential element to be considered in the
process. For DNA extraction, plant material is usually ei-
ther collected fresh and directly subjected to processing
[14], or is stored at 18C, 80C or in liquid nitrogen as
frozen samples before use. Freezing prevents nuclease ac-
tivity that would otherwise degrade the DNA in thawed
tissue [15]. In cases where immediate refrigeration of the
collected samples is not possible, as in field samples or
distant locations, the tissue can be stored in solutions like
saturated cetyltrimethylammonium bromide (CTAB)-
2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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NaCl-NaN3 [16], or dried using materials such as silica gel
and then stored to avoid contamination due to moisture
[17]. Samples stored in the form of lyophilized tissue are a
much better choice as they can be stored for several years
with little loss of DNA quality [18]. However, many isola-
tion methods that have used dried tissues, herbarium
samples or even much degraded material have also been
published [1923].
2.1.3 Homogenization
In plant cells, the presence of a rigid polysaccharide cell
wall makes the disruption of the tissues a little complicat-
ed, taking about 3070% of the total time required for
sample processing. The standard physical lysis proce-
dures for release of DNA become less productive in this
case, as the same physical forces that are required to
break the cell wall are sufficient to shear the high molec-
ular weight DNA [1]. It becomes more difficult with peren-
nial plants or tree species with tough leaves, which may
require special grinding procedures [16, 24, 25] to achieve
complete disruption of the tissue to get DNA. Proper ho-
mogenization is necessary as it also reduces viscosity
caused by high molecular weight compounds like com-
plex carbohydrates [26]. A wide variety of physical dis-
ruption methods are used, which include grinding the tis-
sue mechanically using mortar and pestle, steel or glass
rods, glass beads, etc. Grinding the tissue in liquid nitro-
gen using a flash freezing procedure is preferred, largely
as it is rapid, applicable to both soft and hard tissue and
also prevents cross contamination when multiple samples
are being processed [4, 2730]. The prime limitation with
liquid nitrogen is the special storage conditions and high
cost, which makes it unsuitable for less-equipped labora-
tories [31, 32].
Besides the physical disruption methods for tissue ho-
mogenization, convenient chemical disruption methods
are also available. This approach though expensive, cir-
cumvents the possibility of DNA getting sheared due to
mechanical force applied. Chemical disruptors mainly in-
clude hydrolyzing enzymes such as cellulases, pectinases
or cell wall macerases to dissolve the cell wall. A cocktail
of various carbohydrases has also been found suitable for
chemical digestion in various species that include Ilex
aquifolium, Vitis vinifera, Humulus lupulus, Geranium sp.,
etc. [33]. A slightly different approach of using xanthate-
forming compounds, such as sodium/potassium ethyl
xanthogenate, has also been reported in this regard [34].
These compounds form water-soluble polysaccharide
xanthates with the hydroxyl groups of cell wall polysac-
charides and dissolve it. Without its cell wall, a plant cell
is like any other cell whose membrane can be lysed using
a detergent. This approach is particularly useful for tough
leaves like that of rice,which contain large amounts of cel-
lulose, pectin, silica cells, wax, etc. [6]. However, the
method is reported to yield very low quantities of DNA
[33, 35].
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2.2 Presence of contaminants
The most frequently encountered problems with almost
all the procedures are the presence of contaminating
compounds in the DNA samples, particularly RNA, pro-
teins, polysaccharides, polyphenolics and non-nuclear
DNA. Secondary metabolites amplify the difficulties with
plant cells, particularly when medicinal plants are used as
the source. The compounds that provide the therapeutic
efficacy to the plant may prove to be a major hindrance in
the isolation procedure by binding with the DNA and be-
ing precipitated along with it [36]. Thus, according to the
variety of substances present along with the DNA, proto-
cols need to be tailored according to each plant species
(and sometimes even tissues), making the process of iso-
lation a skill or an art that has to be developed by repeat-
ed practice. Some major contaminants are described be-
low.
2.2.1 Polysaccharides
Polysaccharides are the prime interferers in the DNA iso-
lation procedure as they are undetectable and hard to re-
move. The presence of high amounts of polysaccharides,
as in case of mucilaginous plants such as Sedum telephi-
um [27], makes the tissue homogenate very viscous, and
gives a false indication of presence of high amounts of
DNA. Polysaccharides often co-precipitate with the DNA
and impart a sticky, viscous consistency to it. In this form,
they also interfere with the activity of several biological
enzymes like polymerases, ligases and restriction en-
donucleases [10, 21]. Polysaccharide-like contaminants
can also cause anomalous re-association kinetics [1], and
render the DNA unsuitable for downstream processing.
The contaminated DNA tends to stick in the well during
gel electrophoresis [37, 38].
Use of high concentration (more than 0.5 M) NaCl is
suitable for the removal of polysaccharides from DNA so-
lutions by increasing their solubility in ethanol. The con-
centration of NaCl varies according to the source plant
species [4, 14, 39, 40], e.g., Zidani et al [40] used 1.4 M
NaCl in pearl millet (Pennisetum glaucum) and Aljanabi
and Martinez [14] have applied as high as 6 M NaCl to the
polysaccharide rich wheat (Triticum aestivum) and barley
(Hordium vulgaris). NaCl in combination with the cation-
ic detergent CTAB has also proved to be beneficial in
DNA isolation from polysaccharide-rich plants [1, 41].
CTAB helps in precipitating DNA by forming a complex
with it in a low ionic strength environment, and, at high
salt concentration, it forms insoluble complexes with pro-
teins and most acidic polysaccharides, leaving the nucle-
ic acids in the solution, which then can be easily isolated.
In the parts of plant in which excessive polysaccharide is
present, as in case of tubers and seeds, polysaccharide
binding resins can be applied, which eliminate the con-
tamination. Rogstad et al. [42] employed hydrolytic en-
zymes such as pectinase to digest pectin-like contami-
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Biotechnol. J. 2007, 2, 386392
nants in plants such as Quercus rubra, Castanea dentate,
etc. A simple and effective way can be by diluting the
DNA extracts or washing the homogenate two or three
times with extraction buffer to reduce polysaccharide
contamination [11, 21].
2.2.2 Polyphenols
Perhaps the most frequently encountered problem in the
plant DNA isolation protocols is the degradation of ge-
nomic DNA due to presence of polyphenols. Polyphenols
are extremely variable in their occurrence and type. Dur-
ing cell lyses, polyphenols come out of the vacuoles and
are readily oxidized by cellular oxidases. The oxidized
polyphenols undergo irreversible interactions with nucle-
ic acids and causes enzymatic browning of the DNA pel-
let, thereby rendering it useless for most downstream
processes [4, 7, 9, 13, 29, 4345]. Polyphenolics like
flavonoids, terpenoids and tannins, which all occur very
widely in plant kingdom, when bound to the nucleic acids
cannot be removed by conventional extraction proce-
dures. To deal with the problem of polyphenolic contami-
nation, antioxidants are added to the extraction buffer to
prevent the oxidation of phenols during cell lysis.
Polyvinyl pyrollidone (PVP) and polyvinyl polypyrollidone
(PVPP) are the most commonly used chemicals to elimi-
nate polyphenolics as they act as adsorbents of polyphe-
nols, especially at low pH. They form complexes with the
polyphenolic compounds through hydrogen bonding, al-
lowing the polyphenolics to be separated from the DNA,
thereby reducing their levels in the product [4, 43, 4547].
Either or both compounds may be used in suitable pro-
portions, as demonstrated by Basak et al. [48] in Vigna
mungo and Bambusa balcoa, etc., with good results. PVP
10 is preferred to PVP 40, as the later may sometimes pre-
cipitate with nucleic acid, thereby acting as a contami-
nant [37]. Antioxidants such as -mercaptoethanol, BSA,
sodium azide, ascorbic acid, DTT, sodium sulfite, sodium
iso-ascorbate, etc., along with PVP are commonly used to
deal with problems related to phenolics [8, 17, 28, 35, 37,
39, 49]. -Mercaptoethanol in particular is widely used,
and prevents the polymerization of tannins that hinder
the isolation process in a manner similar to polysaccha-
rides. In plants with high levels of polyphenolics, such as
tomato, grapevine, Arabidopsis, canola, cocoa, etc., the
use of a specific inhibitor of polyphenol oxidases, such as
DIECA is also reported [19, 35, 46, 50]. Permingeat et al.
[46] used glucose as a reducing agent in cotton (Gossyp-
ium hirsutum) to avoid contamination and browning by
polyphenolics.
2.2.3 Proteins and RNA
A substantial amount of proteins and RNA may get pre-
cipitated with DNA. RNA is usually purged using DNase-
free pancreatic RNase; if this is unavailable, lithium chlo-
ride can be effectively used for the purpose [27, 31]. Pro-
teins are generally removed using chemicals like SDS,
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Biotechnol. J. 2007, 2, 386392
DTT and -mercaptoethanol, which destroy the structur-
al organization of proteins. Protein-hydrolyzing enzymes
like proteinase K are also suitable for the purpose as they
break a variety of peptide bonds and rapidly inactivate
DNases and RNases in cell lysates, which facilitates the
isolation of high molecular weight DNA. Grinding of plant
materials in an SDS extraction buffer containing pro-
teinase K may be required in case of protein-rich tissues,
e.g., seeds [14, 51]. Conventionally proteins are removed
by extracting with organic solvents like phenol and chlo-
roform. The major drawback of this procedure are the
caustic and toxic properties of phenol and chloroform, in
addition to a considerable loss of the sample that is seen
after phenol extraction. Thomas et al. [52] suggested use
of gel-barrier tubes for safer phenol-chloroform extraction.
A large number of protocols do not recommend the use of
phenol [6, 14, 53], or advice avoiding it when extracting
DNA from phenolic-rich plant species. In cases of source
material with a high lipid content, like oils seeds, defat-
ting the material before DNA isolation has proved benefi-
cial, as lipids form a visible layer over the homogenate and
cause difficulties in further processing [54].
2.2.4 Non-nuclear DNA
In addition to the contaminants described above, chloro-
plast and mitochondrial DNA are also considered as con-
taminants in some key molecular biology applications,
like genomic library construction and screening, DNA hy-
bridization studies and research on DNA renaturation,
etc., where only pure nuclear DNA can be used. The
chloroplast in particular is considered a major contami-
nant in processes involving plants. To contend with this
problem, nuclei can be isolated before extraction. In re-
calcitrant plant species like cotton, grape, etc., DNA iso-
lation through nuclei isolation minimizes the problems of
contamination due to high polysaccharide and polyphe-
nolic levels in the cells.
Nuclei can be isolated from the plant cells by homo-
genizing the plant tissue in a suitable isolation buffer con-
taining osmoprotectants, nuclear membrane stabilizers,
and sometimes a non-ionic detergent Triton X-100 [5, 7,
12, 15, 29]. The presence of osmoprotectants like glucose,
and sucrose, and membrane stabilizers like spermine,
spermidine, and polylysine in the homogenization buffer
stabilize the nuclear membrane, thereby facilitating the
isolation of intact nuclei by centrifugation. Addition of Tri-
ton X-100 specifically lyses chloroplasts and mitochon-
dria, leaving the nuclei intact [7, 12, 15, 50], which can
then be isolated by centrifugation. The nuclear pellet ob-
tained after centrifugation is then lysed using nuclei lysis
buffer and the DNA obtained by a cesium chloride gradi-
ent ultracentrifugation method [15, 50]. This technique
yields high-quality DNA, but has the prominent disad-
vantages of being expensive, time consuming and incon-
venient. Most of the recent nuclei isolation protocols do
not involve cesium chloride centrifugations, but use chlo-
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roform extraction and ethanol precipitation after nuclear
lysis to obtain pure nuclear DNA.
3 DNA isolation kits
Procedures involving isolation of nuclei or cesium chlo-
ride gradient centrifugations may yield high-quality DNA
but is very time consuming. Thus, a rapid and less ex-
pensive method that gives quality DNA is preferred by the
majority of molecular biologists today [55]. A remarkable
advancement in this approach is the availability of com-
mercial DNA isolation kits that are easy to use and pro-
vide fast and better results. These are equipped with me-
chanical cell disruptors (for fine grounding of tissue), lysis
buffers, RNase, etc. Proteins and polysaccharides are re-
moved by salt precipitation and centrifugation ([16, 25]
and www.qiagen.com). The majority of these kits use
high-quality resins or disposable chromatographic
columns, which have different elution protocols ([56] and
www.qiagen.com). The resins rely on mixed ion-ex-
change/adsorption interaction for purification, use silica
gel to which the DNA binds in a reversible manner in
presence of chaotropic salts and elutes out in the column
effluent when rehydrated by washing with aqueous
buffers [57]. Large scale automatable DNA mini-prep kits
are offered by several companies: Qiagen, Promega,
Epibio, Cartagen, Roche, Epicentre biotechnologies, etc.
Thus, pure DNA can be obtained within approximately
1 h using these ready to use kits, but most of these are not
cost effective enough to be used on a general laboratory
scale. As a cheaper alternative, the resins can be pur-
chased in bulk and packed in ordinary glass columns or
syringes for immediate use in laboratories [10, 11, 58]. Re-
sults using kits can, however, also be inefficient at times
when handling plants with high polysaccharide or high
polyphenolic content like peanut (Arachis hypogea) and
members of Asteraceae (Cichorioideae) species, e.g., Ci-
chorium intybus, Taraxacum officinale and Lactuca sati-
va [8, 11].
4 Assessment of DNA quality and yield
A range of methods is available to assess the quality of the
isolated DNA, which include gel electrophoresis, spectro-
metric analysis, restriction digestion, PCR amplification,
and chromatographic techniques. Nevertheless, the pri-
mary evaluation comes from the viscosity and clearness
of the DNA solutions. The most frequently used technique
for quality assessment is spectrometric analysis of nucle-
ic acids. Being rapid, easy and comparatively cheap, it is
quite often the method of choice. The ratio of absorbance
at 260/280 nm is 1.8 for a pure DNA sample and a decrease
indicates contamination by (largely) proteins, while the
presence of RNA increases the ratio (above 2.0) [9, 57].
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Plant DNA preparations have a realistic chance to have
impurities of polysaccharides and polyphenolics, which
can be detected by taking readings at 230 nm and 270 nm.
The ratio A260/A230 must be greater than 1.8 and ratio
A260/A270 should lie between 1.2 and 1.3 in DNA prepara-
tions free from contamination of polysaccharides and
polyphenolics, respectively [7, 20, 31, 35]. Agarose gels
can also give an indication of impurities such as salt, RNA
and carbohydrates. The DNA can be subjected to restric-
tion enzyme digestion as restrictable DNA is considered
significantly pure DNA. Apart from this, HPLC chro-
matography can also be employed to analyze DNA solu-
tions [9], where no prominent peaks at 230, 270, and
280 nm and a prominent peak at 260 nm in the spectra
would indicate contamination-free DNA. The yield of
DNA can be determined by spectrophotometric analysis,
using the assumption that an absorbance of 1 at 260 nm
corresponds to 50 g double-stranded DNA in 1 mL. How-
ever, as DNA and RNA both absorb strongly at 260 nm and
the absorbance enhances with the presence of single-
stranded DNA, this may give a false interpretation of the
quantity. The spectrophotometric technique is thus not
considered very accurate for quantifying DNA. Flourimet-
ric estimation of DNA yield using the dye Hoechst 33258
is considered a better option. Additionally, estimates can
be obtained using agarose gels with different concentra-
tions of standard loaded in separate wells; here the as-
sumption is that their band intensities are proportional to
the amount of sample loaded.
5 Conclusions
Considering the various factors involved, the thumb rule
remains use what works for the plant of interest. Any
protocol, whether it involves lengthy cesium chloride cen-
trifugations, nuclei isolation, expensive reagents, organic
solvents or DNA isolation kits, proves worthy only when it
yields uncontaminated DNA, preferably in a short time.
Researchers by and large pursue the basic protocols [13],
and incorporate modifications according to their require-
ments. When struggling with secondary metabolites, the
key modifications usually involve varying the concentra-
tions of reagents like CTAB, NaCl, PVP and other antioxi-
dants, hydrolyzing enzymes, column purifications etc.,
which may again vary with the plant, and even tissues
within the plant. Although there are examples of proce-
dures that have been applied to a variety of plant species
[14, 19, 31, 33], the chemical heterogeneity and diversity
of plant systems renders it very difficult for a single
method to be employed commonly for isolating pure, re-
strictable plant genomic DNA. Hence, there is an obliga-
tion to develop a potential broad-spectrum method that is
cost effective, avoids use of hazardous reagents, is less
time consuming and, most importantly, could be relevant
to an extensive range of plant sources. Designing of such
390 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Biotechnol. J. 2007, 2, 386392
a method would require an artistic attitude with a scien-
tific approach, which will help in making the targets fair-
ly convenient for plant molecular biology workers.
One of the authors (A.V.) wishes to thank Council of Sci-
entific and Industrial Research, New Delhi, India for fi-
nancial support.
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Neeta Shrivastava has post graduate
and doctorate degrees in plant sci-
ences. Her research areas is plant
biotechnology, molecular biology and
phytochemistry and pharmacognosy.
Her research interests also include phy-
topharmacology. Presently she works
as a plant scientist in BV Patel PERD
Centre, Ahmedabad. She has 37 publi-
cations, which include research articles,
invited reviews, chapters in book and monographs. She has guided
many students for post graduate dissertation. She has three students
working with her for doctoral program.
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