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Direct extraction of genomic DNA from maize with aqueous ionic liquid buffer
systems for applications in genetically modified organisms analysis

Article in Analytical and Bioanalytical Chemistry · November 2014

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Anal Bioanal Chem
DOI 10.1007/s00216-014-8204-y
PAPER IN FOREFRONT
Direct extraction of genomic DNA from maize with aqueous ionic
liquid buffer systems for applications in genetically modified
organisms analysis
Eric Gonzalez García & Anna K. Ressmann &
Peter Gaertner & Ronald Zirbs & Robert L. Mach &
Rudolf Krska & Katharina Bica & Kurt Brunner
Received: 23 July 2014 / Revised: 10 September 2014 / Accepted: 18 September 2014
# Springer-Verlag Berlin Heidelberg 2014
Abstract To date, the extraction of genomic DNA is consid-
ered a bottleneck in the process of genetically modified or-
ganisms (GMOs) detection. Conventional DNA isolation
methods are associated with long extraction times and multi-
ple pipetting and centrifugation steps, which makes the entire
procedure not only tedious and complicated but also prone to
sample cross-contamination. In recent times, ionic liquids
have emerged as innovative solvents for biomass processing,
due to their outstanding properties for dissolution of biomass
and biopolymers. In this study, a novel, easily applicable, and
time-efficient method for the direct extraction of genomic
Eric Gonzalez García and Anna K. Ressmann contributed equally to the
here presented study
Electronic supplementary material The online version of this article
(doi:10.1007/s00216-014-8204-y) contains supplementary material,
which is available to authorized users.
E. Gonzalez García : K. Brunner (*)
IFA-Tulln, Center of Analytical Chemistry, Vienna University of
Technology, Konrad Lorenz Str. 20, 3430 Tulln, Austria
e-mail: kurt.brunner@tuwien.ac.at
A. K. Ressmann : P. Gaertner : K. Bica (*)
Institute of Applied Synthetic Chemistry, Vienna University of
Technology, Getreidemarkt 9/163, 1060 Vienna, Austria
e-mail: katharina.bica@tuwien.ac.at
R. Zirbs
BOKU - University of Natural Resources and Life Sciences Vienna,
Laboratory for Bioinspired Materials, Department of
NanoBiotechnology, Muthgasse 11, 1190 Vienna, Austria
R. L. Mach
Institute for Chemical Engineering, Vienna University of
Technology, Gumpendorfer Str. 1a, 1060 Vienna, Austria
R. Krska
University of Natural Resources and Life Sciences, Department
IFA-Tulln, Konrad Lorenz Str. 20, 3430 Tulln, Austria
DNA from biomass based on aqueous-ionic liquid solutions
was developed. The straightforward protocol relies on extrac-
tion of maize in a 10 % solution of ionic liquids in aqueous
phosphate buffer for 5 min at room temperature, followed by a
denaturation step at 95 °C for 10 min and a simple filtration to
remove residual biopolymers. A set of 22 ionic liquids was
tested in a buffer system and 1-ethyl-3-methylimidazolium
dimethylphosphate, as well as the environmentally benign
choline formate, were identified as ideal candidates. With this
strategy, the quality of the genomic DNA extracted was sig-
nificantly improved and the extraction protocol was notably
simplified compared with a well-established method.
Keywords Maize . Genomic DNA . Ionic liquids . Biomass
dissolution . Genetically modified organisms
Introduction
Ever since the production of the first genetically modified
(GM) crop in 1983, the remarkable progress seen in biotech-
nology has taken farmers to introduce GM crops with charac-
teristics of interest into their agricultural practices. The culti-
vation of these crops has been increasing at an annual rate of
6 %, up to 170 million hectares of biotech crops in 2012 [1].
At the same time, controversial discussions about the potential
risks that genetically modified organisms (GMOs) might exert
on the environment and the human health through foods have
also intensified. In order to deal with growing public concern
about GMOs, regulations have been implemented in numer-
ous countries to enforce their management and labeling. The
European Union adopted the regulation (EC) No. 1829/2003
to set a limit of 0.9 % for food containing authorized GMOs;
products exceeding this limit have to be labeled accordingly
[2]. Usually, methods based on the polymerase chain reaction

(PCR) are applied to quantify the GMO content of a sample
and validated procedures have been developed for all EU-
authorized crops, such as maize, soy, and canola [3]. Distinct
methods are available for the event-, construct-, or element-
specific detection of GMOs. However, due to a growing
number of GM-events, the search for modified crops is time-
consuming and their procedures are complex. To overcome
this drawback, only recently several approaches using simple
isothermal amplification reactions have been published [4–6],
including the idea of performing on-site screening tests for the
commonly used 35S promoter. However, these publications
only focused on the development of simple amplification
methods; none of them included a real simplification of the
DNA extraction procedure. In this sense, common available
methods are either based on the surfactant
cetyltrimethylammonium bromide (CTAB) or on commercial
kits, which are time-consuming and tedious, as they include
numerous pipetting and centrifugation steps, which can limit
on-site testing. All of these handling steps are prone to errors
and, furthermore, they bear a certain risk of cross-contamina-
tions. Additionally, the CTAB method uses organic solvents
such as chloroform and isoamylalcohol [7], which are envi-
ronmentally problematic and might eventually become re-
stricted. In addition, all currently used extraction protocols
require specific laboratory instruments, such as centrifuges,
which can limit its application for on-site testing.
In recent times, ionic liquids (ILs, salts melting at a tem-
perature lower than 100 °C) have attracted the attention of the
scientific community. Based on their outstanding dissolution
properties, they have emerged as innovative solvents for bio-
mass processing. In the past years, different biopolymers,
including wood, cellulose, lignin, chitin, and others were
successfully dissolved and processed in ILs [8]. This powerful
dissolution enables access to valuable ingredients embedded
in the biopolymer matrix and thus allows enhanced extraction
and isolation of active ingredients. Several examples of valu-
able ingredient extraction with ILs have been reported [9–13].
Furthermore, the use of ILs for the isolation of valuable
essential oils allowed not only a simple isolation process for
the pure volatile fragrance components but also a complete
recovery of the IL [14–16]. However, the high price of ILs is
often considered to be a major drawback and, therefore, IL-
water mixtures were successfully applied for the extraction of
valuable ingredients [17–20].
Apart from the use of ILs for the dissolution of biomass,
ILs are also suitable solvents for proteins [21–23], and several
publications showed the superior stabilization of proteins in
IL-aqueous systems compared with conventional buffer solu-
tions [23–25]. The biocompatible IL choline dihydrogen
phosphate tremendously increased the stability of cytochrome
c to more than 6 mo, whereas denaturation in Tris buffer
solution was observed after 2 wk only [23]. Thermal denatur-
ation of ribonuclease A around 60 °C occurred with increasing
E.G. García et al.
hydrophobicity of ILs [26, 27]. Interestingly, Weingaertner
et al. described in 2012 [28] that the choline cation—which
is classified as a denaturation agent—was able to stabilize hen
eggwhite lysozyme and α-lactalbumin.
In contrast, the stability of DNA in ILs remained unknown
until the group of MacFarlane [29] focused on long-term
stability of DNA in various choline based IL-water solutions.
DNA analysis from salmon via circular dichroism spectra
revealed that the double-helical structure of DNA dissolved
in choline lactate was still present after 6 mo of storage at
room temperature, whereas the helical structure was lost after
1 mo in water. Remarkably, DNA samples in the IL choline
dihydrogen phosphate (CDP) (CDP/H2O=50:50) could be
stored for 1 y at room temperature without structural loss,
whereas dissolution of DNA in conventional solvents such as
methanol, DMSO, or formamide resulted in rapid denatur-
ation of DNA. In 2012, the group of Sugimoto [30, 31]
evaluated the effect of ILs on the base pair stability. In contrast
to classical buffer systems, A-T base pairs were more stable
than G-C pairs in hydrated choline dihydrogen phosphate.
Apart from this stabilization effect, ILs were applied to DNA
separation or purification. In 2007, Wang et al. [32] extracted
double-stranded (ds) DNA with 1-butyl-3-methylimidazolium
hexafluorophosphate from aqueous solution. The interactions
between the cationic imidazolium groups and the phosphate
groups in DNA were held responsible for the efficient extrac-
tion, and proteins and metal species did not interfere with the
separation and purification process. In turn, Li et al. [33]
proposed a set of novel ILs for in situ dispersive liquid–liquid
microextraction of DNA with high extraction efficiency. An
aqueous DNA sample was spiked with a complex matrix (e.g.,
proteins or metal ions) before addition of a hydrophilic IL. The
addition of lithium bis(trifluoromethanesulphonyl)imide
LiN(Tf)2 resulted in the formation of a biphasic system with
DNA going into one phase, and thereby a purification effect
could be obtained; best results were obtained with the ILs
1-(1,2-dihydroxypropyl)-3-hexadecylimidazolium bromide
and N,N-dodecyl-N-methyl-d-glucaminium bromide. Only re-
cently, a patent disclosed a method for enhancing efficiency and
sensitivity in nucleic acid amplification from biological mate-
rials such as mucus, blood, stool, and tissue samples using the
IL 1-butyl-3-methylimidazolium tetrafluoroborate [34].
Amplification of DNA in PCR analysis was also observed for
two different gene fragments, A (266 bp; 80 % GC content) and
B (501 bp; 55 % GC content) of Streptomyces coelicolor
genomic DNA. A bicyclic imidazolium bromide (1-butyl-2,3-
tetramethyleneimidazolium bromide) was found to be the best
promoter, whereas commonly used enhancing reagents such as
DMSO and betaine were found to be completely ineffective
[35].
The combination of exceptional dissolution properties and
stabilizing effects for DNA makes ILs ideally suited for en-
hanced and efficient extraction of DNA from crude biomass.
Direct extraction of genomic DNA from maize
Since ILs are also known to promote PCR amplification of
DNA, they are predestined for challenging bioanalytical prob-
lems. Here, we combine these aspects and present a novel,
fast, and efficient strategy for the on-site detection of geneti-
cally modified maize, relying on aqueous ILs after testing
several ILs of potential interest for such a purpose.
Material and methods
Maize samples and chemicals
Maize variety RWA38 was kindly provided by the Institute
of Biotechnology in Plant Production at IFA-Tulln, Austria
and ground either to a powder of gross particle size with an
Oster blender (Boca Raton, FL, USA) or to a fine powder
with a Retsch ball mill MM301 (Haan, Germany). The
particle size distribution of both gross and fine powders
can be found in the Electronic Supplementary Material
(ESM), Figure S1. Maize variety Bt-11 was bought from
the Institute for Reference Materials and Measurements of
the European Union. Samples were stored at 4 °C when
received until usage.
Commercially available reagents and solvents were
used as received from Sigma Aldrich unless otherwise
specified. O,O-Diethyl dithiophosphate was bought from
Merck. 1-Ethyl-3-methylimidazolium acetate
([C 2 mim]OAc), 1-ethyl-3-methylimidazolium chloride
([C2mim]Cl), and choline dibutyl phosphate were pur-
chased from Iolitec (Heilbronn, Germany). All other ILs
were prepared as previously reported, and analytical data
were in accordance with literature. Detailed information on
synthesis of ILs and extraction procedures can be found in
the ESM. ILs were dried for 24–48 h at 80 °C or at room
temperature and 0.01 mbar with stirring before use and
were stored under argon. Double distilled deionized water
was obtained from a Millipore Milli-Q water purification
system (Millipore, USA).
SEM pictures
Scanning electron microscopy (SEM) pictures were taken
with a FEI Inspect F50 at 15 kV. All samples were coated
with a 3.5 nm thick gold-layer using a Leica Cool Sputter
Coater EM SCD005.
Extraction procedures
Extraction procedure using pure ILs
Maize powder (100 mg) was stirred in 900 mg of an IL for
15 min or 1 h at room temperature or 80 °C. The solution
was diluted to 5 mL with Milli-Q water, denaturated at
95 °C for 10 min, centrifuged, and the supernatant ana-
lyzed via real-time PCR. All experiments were carried out
in duplicate or triplicate. The detailed protocol can be
found in the ESM.
Extraction procedure using IL/phosphate buffer
One thousand μL of a solution of IL in buffer and
100 mg maize were stirred for a defined time at room
temperature or 80 °C. The mixture was denaturated at
95 °C for 10 min, centrifuged, and the supernatant
analyzed via real-time PCR. All experiments were car-
ried out in triplicate and performed with or without
5 μL proteinase K (prot K) per assay. The detailed
protocol can be found in the ESM.
CTAB extraction protocol
The extraction was carried out based on the maize DNA
isolation method proposed by the European Union
Reference Laboratory for GM Food and Feed [6] (see ESM
for more details).
Real-time PCR assays
The real-time PCR assays were performed on a RotorGene-
Q cycler (Qiagen, Hilden, Germany). For each reaction,
2 μL template DNA was mixed with 13 μL reaction mix
containing 7.5 μL Kapa Probe Fast (PeqLab, Erlangen,
Germany), 4.78 μL sterile and nuclease-free water, and
0.24 μL for each of the forward and reverse primers
(Sigma-Aldrich, St. Louis, MO, USA) and for the JOE-
labeled probe (Eurofins MWG Operon, Ebersberg,
Germany) as well (each at 6.25 pmol/μL). The Cq values
obtained by quantitative PCR were used to assess the
amount of amplifiable DNA.
The ADH1 gene encoding for the alcohol dehydrogenase,
which is present in both GM (Bt-11) and non-GM maize
samples (RWA38), was used for the determination of the total
amount of maize [7]. In contrast, the cauliflower mosaic virus
(CaMV) promoter P35S was utilized for the detection of the
genetically modified sequences in maize. The P35S assay was
used only when comparing the extraction performances for
GM maize certified reference material (maize variety Bt-11)
between the CTAB method and the here proposed IL-based
method of extraction. The rest of the experiments were carried
out with the RWA38 maize variety. The sequences of primers
and probes for the ADH1 and the P35S assay were previously
published [8].
In addition, calibration curves with six points following 2-
fold dilutions were made and linearity and PCR efficiency
were calculated in order to assess the presence of qPCR

inhibitors that might be coextracted. qPCR efficiency was
calculated as follows:
Efficiency ¼ 10ð slope Þ −1
−1
where slope is the slope of the linear regression in the
calibration curve.
DNA concentrations of the extracts were photometrically
measured in a NanoVue Plus spectrophotometer (GE
Healthcare, Little Chalfont, USA). The yield was calculated
as:
Yield ¼ 10cðDNAÞ ⋅ V
where c(DNA) is the DNA concentration photometrically
measured [ng/μL] and V is the total sample volume [μL].
Yield is expressed as μg DNA/g sample.
Results and discussion
The protocol for the IL-based extraction of DNA was devel-
oped in several steps. First, the whole procedure was opti-
mized with non-GM maize (RWA38) and ADH1 was applied
to assess the extraction efficiency. Finally, the certified refer-
ence material for analysis of the GM maize Bt-11 was used to
allow a comparison of the new method with the most widely
applied CTAB-based extraction method for maize from the
European Union Reference Laboratories.
Maize extraction with IL-water mixtures
In order to develop a new method for the efficient extraction
of genomic DNA from maize powder, the role of pure ILs
as extraction and dissolution media followed by a dilution
with water was investigated. Initially, focus was put on ILs
that have been successfully applied for biomass dissolution
[8] (e.g., [C2mim]OAc, [C4mim]Cl, [C2mim]Me2PO4, and
the environmentally benign choline lactate ([chol]lac),
which has already been shown to stabilize DNA [29]. A
10 wt% solution of ground maize powder in IL was stirred
for a certain time and temperature to dissolve biomass and
extract DNA. After being diluted with water to 5 mL to
precipitate biopolymers, the sample was denaturated at
95 °C, centrifuged, and 400 μL from the supernatant was
retrieved and later analyzed via real-time PCR (see ESM for
details).
Among the 32 different ILs tested, a clear trend towards
hydrophilic ILs was found— as to be expected from their
E.G. García et al.
biomass dissolving properties (see ESM Table S1 and
Figure S3). Reasonable extraction yields were observed using
[C2mim]OAc and [C4mim]Cl, but also with protic ILs, such as
N-(2-hydroxyethyl) ammonium lactate, whereas only traces of
DNA were extracted using [C2mim]Me2PO4. In contrast, hy-
drophobic ILs, such as [C2mim] PF6 and [C2mim]NTf2, but
also long-chain imidazolium chlorides, failed to extract rea-
sonable amounts of DNA. In comparison, the two-step addi-
tion of LiN(Tf)2 for the in-situ formation of the hydrophobic
bistriflimide IL [33] turned out to be successful, and a good
extraction performance was observed using [C4DBU]Cl in
combination with LiN(Tf)2. Choline derivatives that can be
classified as environmentally benign ILs with low toxicity
[36] and a biodegradable cation [37] performed particularly
well and were therefore selected and chosen for focused
investigations.
In order to investigate the influence of the carboxylate
anion of choline ILs, the carboxylate chain length was
elongated from formate to acetate, lactate, butyrate
(C4COO–), C6COO–,…etc. to C12COO–. Best results were
obtained using choline hexanoate, and various conditions
such as temperature (25 °C, 80 °C) and stirring time
(15 min, 1 h) were optimized. However, despite these
promising results, problems of reproducibility were ob-
served, especially when choline derivatives were involved.
This might be related to variation in pH, since even traces of
remaining acid from the preparation of IL could depurinate
the DNA. In order to avoid the presence of free acid and
investigate the influence of pH, the synthesis of choline
hexanoate was performed with different molar ratios of
choline hydrogen carbonate to acid (1:0.99, 1:0.95,
1:0.90, 1:0.80). A clear pH dependency of Cq values was
seen (see ESM Figure S4). Changing the pH from 8.0 to 9.0
resulted in a substantial improvement of 1 cycle, corre-
sponding to the double amount of DNA extracted.
However, when the pH value was further changed to 9.5,
the amount of extracted DNA could not be further
increased.
Due to this strong pH dependency, a switch to the
combination of an aqueous IL/buffer system for pH
regulation and to scavenge traces of free acid was
chosen. As seen in previous experiments, the ideal pH
value is in the range of 8.5 to 9.0. Apart from the
benefits of pH control and better reproducibility in
aqueous IL/buffer system, it was seen that a lower
concentration of the IL in the system was advantageous
for the extraction process, as a higher concentration
inhibits the polymerase during the PCR (see ESM
Table S2, entries 1–3). Additionally, the phosphate buff-
er (Na2HPO4/NaH2PO4 buffer, 50 mM, pH = 8.5) en-
hanced the extraction efficiency and, therefore, a com-
bination of ILs with this buffer was a suitable system
for the extraction of DNA from maize powder.
Direct extraction of genomic DNA from maize
Fig. 1 ILs used for the extraction with an IL/buffer system
Definition of best candidates for extraction with an
IL/phosphate buffer system
Based on economic reasons, we initially focused on a 10 %
solution of IL in aqueous media and a decrease of volume
from 5 mL to 1 mL of IL in phosphate buffer. This mixture
was stirred for 10 min at room temperature and denaturated at
95 °C for 10 min to deactivate any possible DNases that might
be coextracted and that can later hinder the TaqMan
Fig. 2 Influence of different ILs on the extraction of DNA from maize:
1 g of a 10 w/v% solution of IL in buffer (1 mL phosphate buffer) and
100 mg maize powder were stirred for 10 min, denaturated at 95 °C,
centrifuged and the supernatant analyzed via real-time PCR. The given
polymerase during the amplification. Afterwards, they were
centrifuged and the supernatant was analyzed via real-time
PCR.
A set of different ILs was tested, including derivatives of
choline, 1-ethyl-3-methylimidazolium, 1-allyl-3-
methylimidazolium cations, as well as aprotic ILs containing an
ethanolammonium or protonated 1,8-diazabicyclo[5.4.0]undec-7-
en (DBU) cation (Fig. 1). Best results were obtained with
[C2mim]fom, [amim]Cl, [C2mim]Me2PO4, and [chol]fom; the
Cq values are for the ADH1 gene of maize. Choline derivatives were
synthesized in a 1:0.9 ratio of choline hydrogen carbonate and the
corresponding acid to avoid the presence of free acid. All experiments
were carried out in triplicate. *No amplification observed

Fig. 3 Optimization of the No prot K/RT
concentration of IL to use in the
system IL-buffer (performed with
36.00
[chol]fom 1:0.9). Analyses were
carried out in the presence or ab-
34.00
sence of prot K and at room tem-
perature and 80 °C; 1 g of a de-
32.00
fined concentration of IL in buffer
and 100 mg maize powder were
stirred for 10 min, denaturated at 30.00
95 °C, centrifuged and analyzed
Cq
via real-time PCR. The given Cq 28.00
values are for the ADH1 gene of
maize. All experiments were car- 26.00
ried out in triplicate
24.00
22.00
20.00
1 5
best anion evaluated was the formate. In case of choline carbox-
ylates, synthesis was performed with a slight excess of choline
hydrogen carbonate to avoid the presence of free acid (Fig. 2).
Results showed that when increasing the chain length of
the carboxylate anion, the amount of extracted DNA de-
creased tremendously (from [chol]fom with a yield of
1094.52±65.16 μg DNA/g sample to [chol]oct with 233±
48.68 μg DNA/g sample or [chol]lac with 594±80.24 μg
DNA/g sample to [chol]dop with 150.96±47.44 μg DNA/g
sample). The previously best performing [chol]hex gave mod-
erate extraction yield (662±44.56 μg DNA/g sample), where-
as only traces of DNA were observed when extracting with
[chol]oct (193±48,68 μg DNA/g sample). A further increase
of the chain length resulted in either no extraction of DNA or
no PCR amplification due to a polymerase inhibition caused
by the IL. In this sense, a more hydrophobic and larger
carboxylate resulted in a less efficient extraction effect of the
IL. Among the choline derivatives, the best performing IL was
the [chol]fom, which was further investigated. However, the
differences and influence of residual acid in IL-buffer systems
were not nearly as pronounced as they were when pure ILs
were applied (see also ESM Table S3 for more details).
In addition, a comparison between the anions from
[C4mim]Bu2PO4 and [C2mim]Me2PO4 showed a difference
of 10 Cq cycles, indicating that a longer chain length in anion
and cation results in higher Cq values. The protic [DBU]fom
performed as well as [chol]fom.
Among the best performing ILs, two lead candidates for
further proceeding were selected: [chol]fom, as it is consid-
ered to be environmentally friendly, and [C2mim]Me2PO4,
based on its previously reported biomass dissolving proper-
ties. Their usage confirms their potential in gDNA iso-
lation from complex matrices, making them ideal for the
E.G. García et al.
Prot K/RT No prot K/80 °C Prot K/80 °C
10 25
IL in buffer (w/v %)
development of rapid methods for DNA extraction with
on-site applications.
Optimization of extraction parameters
With [chol]fom as a promising candidate, we further investi-
gated the influence of different extraction conditions. To initial-
ly define the optimal concentration of IL in phosphate buffer,
experiments were carried out at room temperature and 80 °C
with 10 min stirring. Moreover, the influence of the addition of
prot K (5 μL, 20 mg/mL) was evaluated to see whether the
digestion of proteins and enzymes present in the maize sample
could improve the extraction and later the amplification of
DNA (Fig. 3). At low concentrations of IL in buffer (1 w/v%,
5 w/v%) and the pure buffer (as a blank), an influence of prot K
and temperature on the amplification was observed. Using a
1 w/v% solution of IL in buffer without the addition of prot K
gave no amplification at room temperature, whereas low
amounts of DNA were extracted when performing the reaction
at 80 °C (209.2±37.92 μg DNA/g sample). When increasing
the temperature to 80 °C, a significant improvement of 10 cycles
of the Cq value was obtained with the addition of prot K. With a
higher concentration of IL (5 w/v%), amplification for all
conditions was observed. However, with higher temperature
and the presence of prot K, the amount of extracted DNA
increased (1216±60.76 μg DNA/g sample) compared with
room temperature experiments (968±76.6 μg DNA/g sample)
and extractions without prot K (592±56.96 μg DNA/g sample).
A further increase of the concentration to a 10 % solution of IL
in buffer resulted in the improvement of the Cq values obtained.
At room temperature, no significant influence of prot K was
observed, whereas at 80 °C the presence of prot K approxi-
mately doubled the amount of DNA extracted (1176±52.72 μg
Direct extraction of genomic DNA from maize

Table 1 Optimization of the denaturation step from the extraction pro-
tocol using 1 ml of a 10 w/v% solution of [C2mim]Me2PO4 in phosphate
buffer and 100 mg maize powder
Extraction conditions Cq ADH1±STD
5 min stirring, 10 min denaturation 23.86±0.22
0 min, 10 min denaturation 24.69±0.16
5 min stirring, 10 min denaturation, syringe filtration 24.86±0.36
5 min, 5 min denaturation 25.38±0.48
10 m stirring, 0 min denaturation 31.87±1.06
Buffer, 5 min stirring, 10 min denaturation 29.79±0.62
DNA/g sample) compared with the absence of prot K (660±
88 μg DNA/g sample). Higher temperature favored the extrac-
tion. In contrast, when using even higher concentrations of IL in
buffer like 50 w/v% or 75 w/v%, no amplification was obtained
with or without the addition of prot K at both temperatures
studied, which might be caused by a salting out process induced
by the IL. Although the amount of DNA extracted using 80 °C
was increased, the work at room temperature was chosen for
potential on-site applications of the method.
In addition, experiments were also carried out to see how
[C2mim]Me2PO4 at a concentration of 10 w/v% in phosphate
buffer would perform when extracting at room temperature
and 80 °C, with or without the addition of prot K (results not
shown). In this case, it was seen that the Cq values were better
when working without prot K at room temperature, whereas
no significant difference was seen in extractions at 80 °C with
or without prot K. A minor improvement in the amount of
extracted DNA was obtained when working at 80 °C (980±
104 μg DNA/g sample at 80 °C and 744±124 μg DNA/g
sample at room temperature). However, having simple on-site
applications in mind, the extractions were further performed at
room temperature.
When screening for the optimal incubation time, extrac-
tions were carried out for 5, 10, 15, 30 and 60 min. No
dependency on time when extracting with both of the ILs
previously selected was found. Therefore, an extraction time
of 5 min was chosen for keeping the protocol short and
efficient.
Simplification of the extraction process
The extraction process was further optimized to see whether
the final incubation step carried out at 95 °C for 10 min could
be modified to facilitate the process. The extraction time was
kept constant at 5 min, whereas the denaturation time was
shortened from 10 to 5 min. Alternatively, the extraction was
performed without denaturation at all. As it can be seen in the
results given in Table 1, a decrease of the denaturation time
from 10 to 5 min resulted in a slight increase of the Cq value,
whereas a difference of eight cycles was obtained when no
denaturation was performed at all. In addition, when
performing just a denaturation step for 10 min, only a minor
increase of the Cq value was achieved. This suggests that the
extraction step and denaturation of co-extracted enzymes can
be combined while incubating at 95 °C for 10 min to further
simplify the process.
Moreover, a further simplification can be achieved using
a syringe filtration over a PTFE membrane (pore size=
0.2 μm) instead of centrifugation. Filtration (Cq=24.86±
0.36) slightly decreased the amount of amplifiable DNA
compared with centrifugation (Cq=23.86±0.22). Due to its
simplicity, the filtration process was used for further
experiments.
The influence of particle size on the Cq values was inves-
tigated next with the non-GM maize variety RWA38. In this
case, the smaller particle size results in a more efficient ex-
traction as demonstrated by applying the ADH1 assay to the
isolated DNA. When using [C2mim]Me2PO4 and [chol]fom,
the same pattern was observed. Better results were obtained
with [C2mim] Me2PO4 compared with [chol]fom (see ESM
Table 4).

Fig. 4 Stability over time of [C2mim]Me2PO4 [chol]fom CTAB

DNA extracted with
[C2mim]Me2PO4, [chol]fom and 31.00
the CTAB method and stored at
29.00
room temperature. All
experiments were carried out in 27.00
triplicate 25.00
Ct

23.00
21.00
19.00
17.00
15.00
0 1 2 3 4 5 6 7 8 9 10
Days
 E.G. García et al.

Fig. 5 SEM pictures of maize powder after extraction with water and IL-phosphate buffer solutions at 2500× magnification; left: Water, middle:
[C2mim]Me2PO4, right: [chol]fom

Stability over time of the DNA extracted with the candidate negative, as the reference materials are certified for Bt-11
ILs while storing the samples at room temperature for 10 d event and not for the P35S promoter.
was also investigated (Fig. 4). Real time PCR measurements In addition, calibration curves were used to check for the
were done every 2 d. Results show that the DNA is stable in presence of potential PCR inhibitors in the extracted gDNA
the IL and that the Cq values are comparable to those obtained for both maize varieties and when extracting with the CTAB
with the CTAB extraction. method and the ILs [C 2 mim]Me 2 PO 4 and [chol]fom
(Table 3). As can be seen, all qPCR assays for the ADH1 gene
have high linearities (R2 =0.99) and efficiencies (>0.90).
Scanning electron microscopy of maize treated with ionic
Furthermore, the efficiency and linearity of the extractions
liquids
carried out with the method developed in this work are com-
parable to those obtained with the CTAB method. However,
To further elucidate the influence of ILs on biomass, a scan-
the efficiency and linearity in the case of the P35S promoter
ning electron microscopy (SEM) image of recovered maize
were lower than the ones from the ADH1 gene.
powder after extraction and denaturation was recorded
(Fig. 5). One hundred mg maize powder was treated with
1000 mg of a 10 w/v% solution of either [C2mim]Me2PO4
Quality of extracted genomic DNA
or [chol]fom in phosphate buffer or 1000 mg water via stirring
for 5 min, denaturation at 95 °C for 10 min, and recovered via
The quality of the extracted genomic DNA by using both
filtration. Despite the fact that IL-aqueous buffer solutions
[C 2 mim]Me 2 PO 4 and [chol]fom was investigated by
were applied rather than pure IL, some differences in starch
performing an agarose gel electrophoresis. The pattern of
morphology of the recovered maize powder were clearly
the separated DNA fragments was then compared with the
found. This highlights the potential of ILs for biomass disso-
CTAB method (Fig. 6). As can be seen, a smear of great
lution and emphasizes their importance in the extraction pro-
intensity with fragments smaller than 500 bp was obtained
cess of DNA, as this break-up of starch structures could be
in the sample extracted with the CTAB method. These
held responsible for the improved extraction efficiency.
short DNA molecules are typical for the fragmentation of
the DNA during the extraction procedure. No visible sign
Detection of P35S promoter in Bt-11 maize

The suitability of the proposed extraction method for the Table 2 Detection of the P35S promoter in GM maize Bt-11 by the
detection of the P35S promoter from the Cauliflower mosaic CTAB extraction method and when extracting with the [C2mim]Me2PO4.
The given Cq values were obtained with the P35S assay
virus inserted in the genome of the maize event variety Bt-11
was investigated by extracting DNA from Bt-11 certified GM maize content (%) CTAB [C2mim]Me2PO4
reference material (Table 2). As it can be seen, the obtained
Cq values were comparable to those from the CTAB extrac- Cq P35S STD Cq P35S STD
tion. This fact makes the proposed extraction method suitable 0.1 30.49 0.83 30.95 1.95
for the detection of this analytical target in various screening 0.5 27.65 0.25 28.56 0.59
procedures. By a concentration of 0 % of GM maize content, 1 27.00 0.34 27.84 0.66
only traces were found as 50 % of the replicates were negative
for both methods. It could be expected that the results were not All experiments were carried out in triplicate.
Direct extraction of genomic DNA from maize

Table 3 Linearity and qPCR efficiencies of real-time PCR assays of extracted gDNA following the CTAB method and with the ILs [C2mim]Me2PO4
and [chol]fom for the non GM maize variety RWA38 and the GM maize Bt-11. DNA concentration of the extracts as well as the yield is also shown

Maize variety Method of extraction Target c(ng/μl)±STD Yield(μg DNA/g sample)±STD Slope Efficiency R2

RWA38 [C2mim]Me2PO4 ADH1 180±2 721.2±8 –3.52 0.92 0.99

[chol]fom 274±16 1094.5±64 –3.54 0.92 0.99
CTAB 252±1 252.3±5 –3.22 1.04 0.99
Bt-11 [C2mim]Me2PO4 ADH1 174±4 696.7±17 –3.51 0.93 0.99
[chol]fom 130±2 519.3±9 –3.33 0.99 0.99
CTAB 256±2 256.2±9 –3.32 1.00 0.99
Bt-11 [C2mim]Me2PO4 P35S 174±4 696.7±17 –3.77 0.84 0.98
[chol]fom 130±2 519.3±8 –4.05 0.77 0.99
CTAB 256±2 256.2±9 –3.96 0.79 0.99

of fragmentation was seen in the sample extracted with the

ILs.

Conclusions

Within this study, a fast and efficient strategy based on novel

IL technologies for the extraction of DNA directly from a
complex natural matrix was presented for the first time to our
knowledge. Although the use of pure ILs for genomic DNA
extraction was not suitable as reproducibility problems arose,
it was found that combined IL-aqueous buffer systems could
notably increase the amount of extracted DNA compared with
conventional buffer systems. Significant changes in starch
morphology were observed after incubation of maize with
IL-aqueous buffer systems, thereby emphasizing the influence
of ILs in biomass treatment— even when used in aqueous
solutions. Among all systems tested, the IL [C2mim]Me2PO4
in combination with sodium phosphate buffer (50 mM, pH=
8.5) was found to be the most efficient extraction system.
DNA extracted with [C2mim]Me2PO4 had the highest quality
and was stable when stored at room temperature for 10 d.
Optimization of extraction parameters resulted in a simplified
and time-efficient procedure compared with a conventional
CTAB method for the direct extraction of genomic DNA from
maize powder; a time saving of approximately 3 h was
achieved when extracting 10 samples in parallel with both
methods. The method developed here can be considered as a
starting point for future new generation extraction procedures,
which probably are not limited to GMO analysis but can also
be applied to the isolation of DNA from any biological mate-
rial. DNA analysis became mobile during the last decade by
developing portable PCR cyclers and simple amplification
systems like isothermal procedures. The application of ILs
as an extraction facilitator might remove the still existing Fig. 6 Agarose gel electrophoresis for the genomic DNA extracted by
bottleneck of complex, time-consuming and laboratory- using the CTAB method (well 2) and the ILs [C2mim]Me2PO4 (well 3)
dependent DNA isolation protocols. and [chol]fom (well 4). The ladder was loaded into well 1

Acknowledgments Anna K. Ressmann is a recipient of a DOC-
fFORTE-fellowship of the Austrian Academy of Sciences. The work of
Eric Gonzalez García was financially supported by the state of Lower
Austria in cooperation with the European Regional Development Fund.
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S1416/1414 Technology (VUT) in 2011 under
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(2014) Choline ion interactions with DNA atoms explain Gaertner and Dr. K. Bica. In
unique stabilization of A-T base pairs in DNA duplexes: a 2012, she started her PhD thesis
microscopic view. J Phys Chem B 118(2):379–389. doi:10. in the same group and is currently
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c2cc31740k nology (VUT), Austria. Research
36. Nockemann P, Thijs B, Driesen K, Janssen CR, Van Hecke K, in his group is mainly focused on
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(2007) Choline saccharinate and choline acesulfamate: ionic synthesis covering the following
liquids with low toxicities. J Phys Chem B 111(19):5254– three topics: (i) Ionic liquid tech-
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cules for biodegradability. Chem Rev (Washington, DC) 107(6): identification and synthesis. He
2207–2227. doi:10.1021/cr050952t has co-authored about 60 peer-
reviewed journal articles and 1
book chapter and has supervised
about 10 PhD theses and around
20 Master’s theses. Besides these activities, he is currently Dean of
Academic Affairs for Technical Chemistry at VUT and Editor-in-Chief
of “Monatshefte für Chemie/Chemical Monthly.”

Eric Gonzalez García graduated Ronald Zirbs received his PhD

in Food Science at the University in Technical Chemistry at Vienna
of Havana, Cuba in 2006. He is University of Technology in
currently a PhD student in Bio- 2006. He is currently working on
technology at the Vienna Univer- his habilitation on nanoparticle
sity of Technology, Austria. His actuated membrane materials at
research interests focus on the de- the Institute for Biologically in-
velopment of molecular diagnos- spired materials of the University
tic methods to ensure the safety of of Natural Resources and Life
food and feed derived products. Sciences Vienna.
 E.G. García et al.

Robert L Mach studied microbi- Katharina Bica received her

ology and genetics at the Univer- PhD degree in Technical Chemis-
sity of Vienna and received his try at Vienna University of Tech-
PhD in 1993. He specialized in nology in 2006. She accepted a
microbial biotechnology and gene position as research fellow at
technology and rectified his lec- QUILL (The Queen’s University
turer qualification in 1999 for mo- Ionic Liquid Laboratory) in Bel-
lecular biology. To date he pub- fast, UK working with Prof.
lished 124 articles in peer Kenneth R. Seddon and Prof.
reviewed journals, 2 books, 38 Robin D. Rogers. In 2009, she
book chapters and got 10 patents joined the Centre for Catalysis
issued. and Sustainable Chemistry at the
Technical University of Denmark
for a short research stay with Prof.
Rasmus Fehrmann. After
returning to Vienna University of Technology in 2010 for a tenure track
position, she is currently establishing her independent research career
focused on green and sustainable chemistry. Her research interests are
based on clean catalysis in water with a special focus on micellar systems,
on the application of ionic liquids for separations and synthesis as well as
on the recovery of valuable ingredients from waste biomass using ad-
vanced fluid technologies.

Rudolf Krska is Full Professor

for (Bio-)Analytics and Organic
Trace Analysis and is Head of
the Department for Kurt Brunner received his PhD
Agrobiotechnology, IFA‑Tulln degree in Technical Chemistry at
with more than 170 co-workers the Vienna University of Technol-
at the University of Natural Re- ogy. Since 2008 he is the Head of
sources and Life Sciences, Vienna the Molecular Diagnostics work
(BOKU). 2009/2010 he worked group at the IFA-Tulln, Austria.
for one year as A/Chief of Health His current research interest is the
Canada´s Food Research Division development of rapid DNA-based
in Ottawa. He has received 6 sci- methods for the analysis of food
entific awards and is (co‑)author and feed. He focuses on various
of more than 200 SCI publica- innovative approaches including
tions (h-index: 40). His current the application of isothermal am-
research interests are in the area of plant-fungi metabolomics and myco- plification methods and aptamers
toxin determination by novel mass spectrometric in combination with to detect food allergens, GMOs,
DNA- and infrared-based techniques. animal species and mycotoxins.

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