ACTA BIOLOGICA SLOVENICA 
 LJUBLJANA 2007 Vol. 50, [t. 1: 19–29 
Sprejeto (accepted): 2007-11-26
Anion-exchange chromatography using CIM® DEAE disks as a method 
of choice for DNA isolation from lecithin
Ionsko-izmenjevalna kromatografi ja z uporabo CIM® DEAE diskov kot metoda 
za osamitev DNA iz sojinega lecitina
Špela HÖFFERLE1, Ana KRAMAR1, Franc SMREKAR3, Dejan ŠTEBIH2, Jana VOJVODA2,
Jana ŽEL2, Peter RASPOR1
1 University of Ljubljana, Biotechnical Faculty, Department of Food Science and Technology, 
Jamnikarjeva 101, SI-1000 Ljubljana, Slovenia
2 National Institute of Biology, Department of Biotechnology and Systems Biology, Večna pot 111, 
SI-1000 Ljubljana, Slovenia 
3 BIA Separations, Teslova 30, SI-1000 Ljubljana, Slovenia
Abstract. The most important prerequisite for the application of PCR-based methods, among 
them the detection and quantifi cation of genetically modifi ed organisms (GMOs) is the ability to 
extract signifi cant amounts of DNA of adequate quality from the sample under investigation. The 
sample of interest in our work was soybean lecithin with expected low DNA content. The aim of 
this study was to set up a fast and effective HPLC (High Performance Liquid Chromatography) 
method using CIM® (Convective Interaction Media, BIA Separations d.o.o., Ljubljana, Slovenia) 
DEAE (DiEthylAminoEthyl) anion-exchange disk monolithic columns (disks) for the isolation 
of DNA from soybean lecithin samples. As the reference isolation procedure we used CTAB 
(CethylTrimethylAmmonium Bromide) method, which is widely used in GMO detection. It was 
demonstrated, that CIM® DEAE disks allow effi cient isolation of DNA from soybean lecithin. 
Furthermore, in comparison with the CTAB method, the method was less time-consuming and 
reduced the use of some aggressive chemicals. The quality of isolated DNA was tested with 
spectrophotometric analysis, agarose gel electrophoresis and by amplifi cation of soybean speci-
fi c lectin gene with qualitative and real-time PCR. The isolated soybean DNA was of adequate 
quantity and quality for PCR analysis, even though mostly degraded, present in small amounts and 
contaminated with some impurities, among them potential PCR inhibitors. The study expanded 
the applicability of monolithic columns in the isolation of biomolecules from highly processed 
food materials and their potential use for nucleic acids detection. 
Keywords: DNA isolation, detection of GMO, soybean lecithin, chromatography, monoliths, 
CIM, CTAB, PCR, real-time PCR
Introduction
Rapid development in DNA technology, such as PCR, DNA sequencing, DNA cloning techniques, 
DNA restriction analysis and in situ hybridization have created the need for rapid analytical and pre-
parative separation methods for nucleic acids (HUBER 1998). Taking into consideration the difference 
in type, composition and degree of processing of products analyzed, DNA extraction protocols must 
be developed on a case-by-case basis and are of the highest importance (GRYSON & al. 2004). 
20 Acta Biologica Slovenica, 50 (1), 2007
Among possible applications, extraction of signifi cant amounts of recombinant DNA of adequate 
quality, for the use in PCR-based detection methods, represents a crucial step in GMO detection 
(WURZ & al. 1999). Different extraction methods can infl uence the effi ciency of DNA multiplication 
(CANKAR & al. 2006). According to the European legislation, a labelling treshold value of 0,9 % has 
been introduced for food and feed, which is a maximum limit for the accidental and unavoidable 
presence of GMO material per ingredient (Regulation (EC) No. 1829/2003 2003). Labelling is even 
mandatory for derivatives of GMOs, which do not contain DNA or protein resulting from genetic 
modifi cation (GRYSON & al. 2004). 
In 2006, the principle biotech crop, occupying 57 % of global biotech area, continued to be biotech 
soybean (JAMES 2006). Therefore, our main research object was soybean lecithin, which is the most 
important byproduct of the edible soybean oil processing industry because of its functionality and 
wide application in food systems and industrial utility. Lecithin production is an aggressive procedure, 
that yields lecithin as a highly processed hydrophobic matrice (SZUHAJ 1983). 
The most widely and frequently used approaches for the isolation of DNA from plant material 
and plant-derived products are: the CTAB method (LIPP & al. 1999); DNA-binding silica columns in 
form of commercial kits and the combination of both (TERRY & al. 2002). The existing methods for the 
isolation of DNA from foods are not useful for all matrices and cannot always fulfi l the expectations 
regarding the quantity and quality of DNA. More recently, chromatography gained importance in the 
isolation of biomolecules, such as recombinant proteins, peptides, polysaccharides and nucleic acids, 
with the introduction of a novel type of chromatographic supports called monoliths (LEVISON & al. 
1998). CIM® disks allow fast and fl ow-unaffected separation of several biomolecules, including nucleic 
acids (ŠTRANCAR & al. 2002). With the use of anion-exchange chromatography with CIM® disks, the 
separation of oligonucleotides (PODGORNIK & al. 1999); isolation of plasmid DNA from complex ma-
trix, such as bacterial cell lysate (BRANOVIČ & al. 2004); separation of bacterial genomic DNA of size 
up to 200 kbp (BENČINA & al. 2004) and even isolation of DNA from processed foods wih decreased 
DNA content, such as maize and its derivatives (JERMAN & al. 2005) is possible.
The aim of this study was to set up a fast and effective HPLC method using CIM® DEAE disks for 
the isolation of soybean DNA from soybean lecithin samples with expected low DNA content. As the 
reference isolation procedure we used CTAB method, which has been widely used in GMO detection 
(ZIMMERMANN & al. 1998, LIPP & al. 1999, LIPP & al. 2001, GRYSON & al. 2004). The quality and quantity 
of the isolated DNA was tested with spectrophotometric analysis, agarose gel electrophoresis and by 
amplifi cation of soybean specifi c lectin gene with qualitative and real-time PCR. Gene lectin is normally 
used for the calculation of the GMO (Roundup Ready® soybean) content, by dividing the amount of 
transgene with the amount of species specifi c gene (lectin gene) (HOLST-JENSEN & al. 2006). 
Materials and methods
Samples 
Six different soybean lecithin samples produced from soybean of known or unknown origin and 
frequently used in manufacture of foods, were used. The samples were: different liquid soybean leci-
thin (Samples 1, 4, 5, 6 and 7) and granulated soybean lecithin (Sample 8). As control samples with 
expected absence of soybean DNA, powdered wheat lecithin (Sample 2) and spelt lecithin (Sample 3) 
were used, even though in the experiment, very small amounts of soybean DNA were unexpectedly 
detected. The DNA was extracted using two different extraction procedures. Using modifi ed CTAB 
method, Samples 2, 3, 5, 6, 7 and 8 were used with 10 g starting material. Isolation of DNA from 
Sample 1 and 4, using CTAB method was not performed, due to the insuffi cient amounts of these two 
samples. Using anion-exchange chromatography with CIM® DEAE columns, 10 g starting material was 
used for Samples 2, 3, 5, 6 and 7 ; and 6,72 g,  4,3 g and 20 g for Samples 1, 4 and 8 respectively. 
21Š. Höfferle, A. Kramar, F. Smrekar, D. Štebih, J. Vojvoda, J. Žel, P. Raspor: Anion-exchange …
DNA extraction using modifi ed CTAB method
As a reference procedure for extraction of DNA from lecithin samples, we used CTAB method, 
as described by Lipp & al. 1999, modifi ed with the addition of hexane and double distilled water and 
greater amounts of starting material (see section: Samples). We used the following starting amounts 
of chemicals: 10 ml of CTAB-1 buffer (20 g/l CTAB (Acros organics, Germany), 1,4 M NaCl (Mer-
ck, Germany), 0,1 M Tris HCl (Sigma, Germany), 20 mM EDTA (ethylenediaminetetraacetic acid, 
Kemika, Croatia), pH 8), 20 ml of hexane (Merck) and 10 ml of double distilled sterile water. The 
isolations were done in 50 ml-tubes. After the initial incubation at 65 ºC for 30 min, the aqueous 
phase was removed and the treatment with addition of 20 mL of hexane was repeated, followed by 
centrifugation and two successive steps of chloroform addition (20 mL) to the aqueous phase. All the 
following steps were performed as proposed by LIPP & AL. 1999. The fi nal DNA was redissolved in 
100 µl of double distilled sterile water, instead of TE buffer, and was stored at –20 ºC.
Initial lecithin extract preparation for chromatographic isolations
The extracts from lecithin samples were prepared using the fi rst few steps of the formerly de-
scribed modifi ed CTAB method. After the second hexane treatment and centrifugation, the aqueous 
phase was removed and fi ltered through a fi lter of regenerated cellulose with pores of 0,45 µm. The 
extracts were then diluted with double distilled sterile water at the ratio of 3:1 (3 volume units of 
the extract and 1 volume unit of double distilled sterile water), before the separation, as proposed by 
JERMAN & al. (2005).
Separation unit, equipment and mobile phase for chromatography
Separations of DNA from impurities were performed on commercially available anion exchange 
(DEAE) methacrylate-based CIM® disk monolithic column (BIA Separations d.o.o, Ljubljana, Slove-
nia), also used by JERMAN & al. (2005). The disk in the appropriate housing was integrated in HPLC 
system. A gradient HPLC system (Knauer, Germany) was built of two pumps, a dynamic mixing 
chamber, an injection valve with 100 or 500 µl injection loop, a variable-wavelength detector and an 
interface box, connected to computer with Windows operation system and EuroChrom 2000 software. 
In all the experiments, the monitor wavelength of 260 nm was used.
On the basis of the work done by Jerman & al. 2005, the following conditions were chosen for the 
separation of DNA from the samples: loading buffer was 50 mM Tris (Merck), 0,25 M NaCl (Merck) 
and elution buffer 50 mM Tris, 2 M NaCl. The pH of the buffers was adjusted to pH 8, using HCl 
(Merck). Loading time was 6 min, followed by 2 min linear gradient of increasing (0,25 – 2 M) NaCl 
concentration, fl ow rate (Φv) was 1 ml/min. Because of larger amount of impurities in the sample, 
washing with loading buffer was prolonged in comparison to work proposed by JERMAN & al. 2005. 
In experiments, where volume of the sample exceeded injection loop volume, multiple injections 
were performed to avoid loading sample with pumps.
DNA recovery from the HPLC fractions
The fractions with purifi ed DNA were manually collected and DNA was precipitated with absolute 
ethanol, using the procedure proposed by JERMAN & al. (2005), modifi ed by the suspension of DNA 
in 100 µL of double distilled sterile water in the last phase.
22 Acta Biologica Slovenica, 50 (1), 2007
Spectrophotometric analysis
The measurements of the UV absorbance of the isolated samples of DNA at 230, 260 and 280 
nm, for DNA purity determination, were performed on Pharmacia Biotech Ultraspec 2000 UV-VIS 
spectrophotometer in 0,5 ml quartz cuvette. DNA samples (5 µl) were 100 X diluted with double di-
stilled sterile water (495 µl) as proposed by KARCHER (1995). The purity was determined on the basis 
of A260/A280 and A260/A230 ratios (SOMMA 2004).
Qualitative PCR analysis
PCR primer pair GMO3 and GMO4 (Invitrogen, California, USA) were used for amplifi cation 
(Querci & al. 2004). This primer pair is specifi c for the sequence of the single-copy lectin gene and 
yields a PCR product of 118 base pairs. The primers were prepared in a double distilled sterile water. 
The amplifi cation was performed on Bio-Rad PCR i-cycler 3.021 instrument, with the reaction mixture 
and amplifi cation programme proposed by QUERCI & al. (2004). The PCR reaction mixture negative 
control contained 2 µl of  double distilled sterile PCR water instead of the sample. The following control 
samples for the presence of lectin gene were used: positive control – Sample A (DNA from conventional 
soybean, lectin positive) and negative control – Sample B (corn DNA, lectin negative). 
Agarose gel electrophoresis
Agarose gel electrophoresis was performed for determination of the degree of degradation of 
the isolated DNA and for visualization of the PCR products. The size-separation was performed 
with 3 µl of the PCR product solution on a Bio-Rad Power Pac 3000 electrophoresis unit, using 1,5 
% (for determination of the degree of degradation) or 2,5 % agarose gel (for visualization of PCR 
products) in 1 X TAE buffer. 100 bp marker (Fermentas) was used as molecular weight size marker. 
The electrophoresis was running for 180 – 210 min at 60 V. The gel was subsequently evaluated by 
10 min ethidium bromide staining (0,5 µg/ml EtBr) and visualized using Bio-Rad Gel Doc 2000 unit 
and Quantity One software.
Real-time PCR analysis
Real-time PCR was performed for amplifi cation of the sequence of soybean specifi c lectin gene 
and to determine the eventual inhibition effect of PCR reaction in the sample extracts. Reaction was 
performed on an ABI PRISM 7900 HT instrument (Applied Biosystems) as proposed by CANKAR & 
al. 2006, with exception of 10 µl reaction mixture used, containing 1 X Taq Man® Universal PCR 
Master Mix (Applied Biosystems). Taqman® RRS probe (Applied Biosystems, Foster City, CA) was 
labelled with 5`-FAM (6-carboxyfl uoresceine) and 3`-TAMRA (5-carboxytetramethylrhodamine) and 
TM-lectin-F and TM-lectin-R were used as primers (Bundesamt für Gesundheit, (Eds): Schweizerisches 
Lebensmittelbuch. CD-ROM, 311.510, BBL-EDMZ 3003, Bern, Switzerland 2001). Reactions were 
performed in parallels under standard conditions (AbiPrism 7900 HT Sequence detection system and 
SDS enterprise database, User guide, Applied Biosystems 2002). For determination of PCR inhibition, 
10-fold dillutions of the samples were used. Treshold cycle (Ct) values were determined using SDS 
2.1 software (Applied Biosystems) after manual adjustment of the baseline and fl uorescence threshold. 
For determination of PCR inhibition, from the difference in Ct values beetween undiluted and 10-fold 
diluted DNA, slopes of curves and corresponding effi ciencies of PCR reactions were calculated using 
equation E = (10(-1/slope)) – 1(GINZINGER 2002).
23Š. Höfferle, A. Kramar, F. Smrekar, D. Štebih, J. Vojvoda, J. Žel, P. Raspor: Anion-exchange …
Results and discussion
Isolation of DNA from lecithin with anion-exchange chromatography using CIM® DEAE disks 
was tested and effective separation with clear peaks was achieved. As an example, chromatogram 
of Sample 5 is shown in Fig. 1. The peak area depended on the volume of the sample extracts used, 
Figure 1: Chromatogram of DNA isolation from extract of soybean lecithin (Sample 5), with anion-exchange 
chromatography using CIM® DEAE monolithic columns (7,5 mL is correspondent to 10 g of soybean 
lecithin sample; 10 mL load volume after dilution with double distilled sterile water at the ratio of 3:1, 
mobile phase pH  8, fl ow rate Φv = 1 ml/min; using 500 µl inj. loop; 1st peak – elution of impurities, 2nd 
peak – elution of DNA).
Slika 1: Kromatogram osamitve DNA iz ekstrakta sojinega lecitina (vzorec 5) z ionsko-izmenjevalno kromato-
grafi jo z uporabo CIM® DEAE diskov (7,5 mL ekstrakta odgovarja 10 g vzorca sojinega lecitina; naložen 
volumen 10 mL po redčenju z dvakrat destilirano vodo v ramerju 3:1, pH = 8, Φv = 1 ml/min; uporaba 
500 µl inj. zanke; 1. vrh – elucija nečistoč, 2. vrh – elucija DNA).
which was 0,075 – 7,5 ml, and on the amount of the DNA, which was sample dependent (Tab. 1). 
In chromatography, using CIM® DEAE disks, retention of dsDNA is infl uenced by electrostatic 
interactions between the surface potentials created by the positively-charged DEAE groups of an 
anion-exchange stationary phase and the negatively-charged phosphodiester groups of dsDNA (HU-
BER 1998). In comparison with the CTAB method, the steps after the initial extraction, consisting of 
purifi cation with chloroform, few centrifugations and a 60 min precipitation (LIPP & al. 1999), were 
substituted with a single-step chromatographic isolation. The method was less time-consuming and 
reduced the use of some aggressive chemicals. CIM® DEAE disks were previously successfully used 
by JERMAN & al. (2005) for isolation of DNA from corn meal and thermally pre-treated corn meal for 
the preparation of »polenta«. 
In our experiment, the quality of the isolated DNA was tested using: agarose gel electrophoresis, 
spectrophotometric analysis, qualitative PCR and real-time PCR. To test the degradation of DNA, 
agarose gel electrophoresis was applied. The isolated DNA from soybean lecithin samples showed very 
24 Acta Biologica Slovenica, 50 (1), 2007
weak signal or no signal on 1,5 % agarose gel and was highly degraded, for both extraction methods 
used (data not shown). The term »DNA quality« is defi ned as the degree of degradation of DNA and 
by the presence of potential inhibitors of the PCR (MEYER 1999).
Spectrophotometric analysis was performed to determine DNA purity (A260/A280, A260/A230) . As 
shown in Tab. 2, values of the A260/A280 ratios showed very variable and sample dependent results. For 
soybean lecithin samples (Samples 1, 4 and 5-8), ratios were 1,9 or 2, which suggested, that isolation 
Table 1: Total gained extract volumes for different lecithin samples, loading extract volumes for separation and 
corresponding chromatogram peak areas for DNA from lecithin extracts, isolated with anion-exchange 
chromatography using CIM® DEAE disks (detection signal at 260 nm; Samples 1, 4, 5, 6 and 7 – liquid 
soybean lecithin; Sample 8 – granulated soybean lecithin; negative control Samples 2 and 3 – powdered 
wheat and spelt lecithin).
Preglednica 1: Dobljeni volumni ekstraktov vzorcev lecitina, na diske nanešeni volumni ekstraktov in pripadajoče 
površine kromatogramov za DNA, osamljeno iz vzorcev lecitina z ionsko-izmenjevalno kromatografi jno 
z uporabo CIM® DEAE diskov (merjenje absorbance pri 260 nm; vzorci 1, 4, 5, 6 in 7 – tekoči sojin 
lecitin; vzorec 8 – granuliran sojin lecitin; vzorca 2 in 3 za negativno kontrolo – lecitin pšenice in pire 
v prahu).
Sample number 1 2 3 4 5 6 7 8
Total extract 
volume (ml)
4,6 10 6,5 8 7,6 17 7,5 7,1
Loading extract 
volume for 
separation (ml)
4,5 0,075 0,075 7,5 7,5 7,5 7,5 7
Peak area 
(mVmin)
20,8 122,6 513,0 278,8 128,2 88,8 80,2 88,4
Table 2: Comparison of the calculated A260/A280 and A260/A230 ratio values for purity determination of DNA from 
extracts, isolated with anion-exchange chromatography using CIM® DEAE disks or with modifi ed CTAB 
procedure (spectrophotometric analysis of absorption at 230, 260 and 280 nm; Samples 1, 4, 5, 6 and 
7 – liquid soybean lecithin; Sample 8 – granulated soybean lecithin; negative control Samples 2 and 3 
– powdered wheat and spelt lecithin). Remarks: / – analysis for Samples 1 and 4, isolated with CTAB 
method, were not performed.
Preglednica 2: Primerjava izračunanih vrednosti razmerij A260/A280 ter A260/A230 za čistosti DNA, osamljene z 
ionsko-izmenjevalno kromatografi jo z uporabo CIM® DEAE diskov ali z modifi cirano CTAB metodo 
(spektrofotometrične meritve absorbance pri valovnih dolžinah 230, 260 in 280 nm; vzorci 1, 4, 5, 6 in 
7 – tekoči sojin lecitin; vzorec 8 – granuliran sojin lecitin; vzorca 2 in 3 za negativno kontrolo – lecitin 
pšenice in pire v prahu). Opombe: / – analize za vzorce 1 in 4, osamljene po metodi CTAB, niso bile 
opravljene.
CIM® DEAE CTAB
Sample number A260/A280 A260/A230 A260/A280 A260/A230
1 2 0,7 / /
2 1,5 1,3 1,8 2,4
3 1,4 0,2 1,7 0,8
4 2 0,5 / /
5 1,9 1,7 1,7 1
6 1,9 1,7 1,3 1,3
7 2 1,4 2 1
8 2 1,4 2 1
25Š. Höfferle, A. Kramar, F. Smrekar, D. Štebih, J. Vojvoda, J. Žel, P. Raspor: Anion-exchange …
using CIM® DEAE disks satisfactory purifi ed the DNA of proteins (Somma 2004), with exception of 
wheat and spelt lecithin (Samples 2 and 3). The modifi ed CTAB method showed acceptable results 
for Samples 2, 7 and 8 and worse results for Samples 3, 5 and 6. Values of the A260/A230 ratios showed 
a high degree of contamination of DNA by substances, such as carbohydrates, peptides, phenols or 
aromatic compounds (Somma 2004), for almost all lecithin samples analysed, using either CIM® 
DEAE disks or modifi ed CTAB method as isolation methods. The values, with exeption of Sample 
2, isolated using CTAB method, were all under 2,2 (Tab. 2).
Amplifi cation potential of the isolated DNA was tested by amplifi cation of the sequence of lec-
tin gene with qualitative PCR. DNA, isolated from all 8 lecithin samples using CIM® DEAE disks, 
contained amplifi able soybean DNA (Fig. 2). Additionally, lectin gene DNA was also amplifi ed in 
the samples of wheat and spelt lecithin, which suggested that traces of soybean were also present 
in these two samples. The amplifi cation of lectin gene was also performed for the samples, isolated 
using modifi ed CTAB method. The PCR products were visible for 4 out of 6 lecithin samples (data 
not shown). Gryson & al. (2002) showed that degumming in the refi ning process is the most important 
step in transferring DNA from crude soybean oil to the water fraction (lecithin). 
To test the possibility of PCR inhibition, real-time PCR was performed. DNA was assayed for the 
lectin gene at two 10-fold dilutions (undiluted and 10-fold diluted sample). In Tab. 3, the numerical 
results (Ct and E values) for real-time PCR of samples, isolated with modifi ed CTAB method and using 
Figure 2: Agarose gel electrophoresis for visualization of  PCR products (118 bp size) of amplifi cation of soybean 
DNA (GMO3 and GMO4 primers used, specifi c for soybean lectin gene) isolated with anion-exchange 
chromatography using CIM® DEAE disks (lane 1, 4, 5, 6, 7 in 8: Samples 1, 4, 5, 6 and 7 (liquid soybean 
lecithin) and 8 (granulated soybean lecithin); lane 2: Sample 2 for negative control (wheat lecithin); lane 
3: Sample 3 for negative control (spelt lecithin); lane N: PCR reaction mixture negative control; lane +: 
reference Sample A for pozitive control (lectin poz.); lane –: reference Sample B for negative control 
(lectin neg.); M: 100 bp molecular weight size marker).
Slika 2: Agarozna gelska elektroforeza pomnožkov PCR (velikost 118 bp), za odkrivanje sojine DNA v vzorcih 
sojinega lecitina (uporaba začetnih oligonukleotidov GMO3 in GMO4, specifi čnih za sojin lectin gen), 
osamljene z ionsko-izmenjevalno kromatografi jo z uporabo CIM® DEAE diskov (proge 1, 4, 5, 6, 7 in 
8: vzorci 1, 4, 5, 6, 7 (tekoči sojin lecitin) in 8 (granuliran sojin lecitin); proga 2: vzorec 2 za negativno 
kontrolo (lecitin pšenice); proga 3: vzorec 3 za negativno kontrolo (lecitin pire); proga N: neg. kontrola 
PCR reakcijske mešanice; proga + : ref. vzorec A za poz. kontrolo (lectin poz.); proga –: ref. vzorec B 
za neg. kontrolo (lectin neg.); M: molekulski označevalec dolžin pomnožkov 100 bp).
26 Acta Biologica Slovenica, 50 (1), 2007
Isolation method Sample number Ct E (%) Inhibition
CIM® DEAE
1
34,35127
9900# YES#
33,43676
10 X R 33,82257
34,87834
2
32,795334
 364# YES#
33,864666
10 X R 35,62797
34,094425
3
23,401003
 142 YES
23,426117
10 X R 26,106941
25,897501
4
26,325968
 142 YES
26,243254
10 X R 28,75998
28,975714
5
26,04777
 110 NO
26,083706
10 X R 29,08203
29,172789
6
30,621397
 184 YES
30,521042
10 X R 32,649963
32,955513
7
26,466047
  89 NO
26,284533
10 X R 29,997795
29,856054
8
30,247734
 128# YES#
29,934256
10 X R 33,386494
32,515114
CTAB
2
/
/ /
/
10 X R /
/
3
~
~ YES
~
10 X R 25,738462
25,745096
5
31,464403
 105# NO#
31,637342
10 X R 33,677097
35,842133
6
32,044857
  80# NO#
32,421013
10 X R 36,970497
35,285854
7
32,17105
142# YES#
32,099506
10 X R 33,97479
35,506104
8
~
~ YES
~
10 X R 35,271873
34,3671
Table 3: Ct and E values for determination of inhibition of lectin gene amplifi cation with real-time PCR.
Preglednica 3: Ct in E vrednosti za določanje inhibicije pomnoževanja lectin gena s PCR v realnem času.
27Š. Höfferle, A. Kramar, F. Smrekar, D. Štebih, J. Vojvoda, J. Žel, P. Raspor: Anion-exchange …
CIM® DEAE disks, are presented. Inhibitors were present in some of the lecithin samples, isolated 
with modifi ed CTAB method and also in some samples, isolated using CIM® DEAE disks. Because of 
the very low amounts of soybean DNA, the variability of the Ct values between some parallel sample 
dilutions is present. With 10-fold linear dilutions, a 100 % effi ciency of the real-time PCR corresponds 
to a -3,32 slope of a curve (GINZINGER 2002). In our work, we used a slope interval from -4,1 to -2,9, 
corresponding to effi ciencies from approximatelly 75 to 121 % respectively, which suggested an absence 
of PCR inhibitors. Complex plant-derived matrices, usually contain a number of PCR inhibitors, such 
as polysaccharydes, proteines and phenolic compounds. Additionally, some extraction chemicals can 
also act as PCR inhibitors, such as ethanol, EDTA, NaCl, chloroform, isopropanol, CTAB and other. 
Their presence can infl uence the amplifi cation of DNA (ROSSEN & al. 1992). 
Conclusions
The aim of this study was to investigate the applicability of CIM® DEAE disks for the isolation 
of DNA from highly processed hydrophobic matrice, with decreased DNA content – soybean lecithin. 
It was demonstrated, that:
– the anion-exchange chromatography using CIM® DEAE disks, allows effi cient isolation of DNA 
from soybean lecithin. In comparison with the CTAB method, the method was less time-consuming 
and reduced the use of some aggressive chemicals;
– the isolated DNA was of adequate quality for the real-time PCR analysis;
– the soybean DNA was present in small amounts and mostly contaminated with some impurities, 
including potential PCR inhibitors;
The study expanded the applicability of monolithic columns in the isolation of biomolecules from 
highly processed food materials and their potential use for nucleic acids detection.
Povzetek
Prvi in zelo pomemben korak v procesu detekcije nukleinskih kislin, je njena osamitev in čiščenje, 
ki mora zagotoviti DNA primerne količine in kakovosti za nadaljnjo uporabo. Eden od aktualnih pri-
merov uporabe osamljene DNA je pomnoževanje genskih elementov v reakcijah PCR, za zagotavljanje 
natančnega določanja prisotnosti GSO v živilih. Sojin lecitin je eden izmed najbolj pomembnih stranskih 
produktov v industriji procesiranja soje, ki je danes najbolj razširjena gensko spremenjena poljščina. 
Zagotovo je njegova uporaba najpomembnejša in najbolj razširjena v živilski/prehranski industriji. Da 
bi potrošnikom zagotovili sledljivost proizvodov, je odkrivanje morebitno prisotne gensko spremenjene 
soje v tem proizvodu nujno. Cilj našega dela je bil postaviti učinkovito metodo za osamitev DNA soje 
iz sojinega lecitina z ionsko-izmenjevalno kromatografi jo z uporabo CIM® DEAE diskov. V ta namen 
smo skušali osamiti DNA iz osmih vzorcev lecitina z uporabo ionsko-izmenjevalne kromatografi je 
s CIM® DEAE diski, ter ločeno iz šestih vzorcev lecitina z referenčno metodo CTAB. Za negativno 
kontrolo smo uporabili pšenični in pirin lecitin, kjer smo pričakovali odsotnost sojine DNA. Za uspešno 
sta se izkazali tako ionsko-izmenjevalna kromatografi ja z uporabo CIM® DEAE diskov, kot referenčna 
metoda CTAB za izolacijo DNA iz sojinega lecitina. S pomnoževanjem za sojo specifi čnega lectin 
Legende – Legenda:
Ct; E – treshold cycle; effi ciency
/: lectin gene (soybean DNA) not present
~: results lower than the limit of detection
10 X R: 10-fold diluted parallel samples
# – variability of the Ct values between parallel samples, because of low amounts of the isolated soybean DNA.
28 Acta Biologica Slovenica, 50 (1), 2007
gena s kvalitativno reakcijo PCR in reakcijo PCR v realnem času smo preverili kakovost osamljene 
DNA. Prisotnost sojine DNA smo dokazali v vseh vzorcih sojinega lecitina. Osamljena DNA je bila 
torej prisotna v dovoljšnji količini, njena kakovost pa je bila primerna za pomnoževanje v reakciji 
PCR.  Z reakcijo PCR v realnem času smo ugotovili, da so bili v nekaterih ekstraktih analiziranih 
vzorcev lecitina prisotni inhibitorji reakcije PCR. Agarozna gelska elektroforeza je pokazala močno 
razgrajenost osamljene DNA. Spektrofotometične analize za določanje čistosti nukleinskih kislin so 
pokazale, da so v večini analiziranih ekstraktov vzorcev lecitina poleg DNA prisotne tudi številne 
nečistoče. Z ugotovitvami smo razširili področje možne uporabe CIM® DEAE diskov pri osamitvi 
biomolekul iz visoko procesiranih živil in njihovo potencialno uporabo v molekularnih metodah 
določanja nukleinskih kislin.
Acknowledgments
The Slovenian Ministry of Higher Education, Science and Technology is kindly acknowledged for 
the grant No. V1-0879. Mlinotest d.d. (Ajdovščina, Slovenia), Žito Gorenjka d.d. (Lesce, Slovenia), 
Krka d.d. (Novo mesto, Slovenia), Brenntag d.o.o. (Ljubljana, Slovenia) and National Institute of 
Biology (Ljubljana, Slovenija) are kindly aknowledged for providing the samples. Š.H. would like 
to express her thank to Biotechnical Faculty (University od Ljubljana, Slovenia) for the opportunity 
to prepare her diploma thesis.
Literature
AbiPrism 7900 HT Sequence detection system and SDS enterprise database, User guide 2002. Applied 
Biosystems.
BENČINA M., A. PODGORNIK & A. ŠTRANCAR 2004: Characterization of methacrylate monoliths for 
purifi cation of DNA molecules. Journal of Separation Science 27 (10–11): 801–810.
BRANOVIČ K., D. FORČIČ, J. IVANČIČ, A. ŠTRANCAR, M. BARUT, T. KOŠUTIČ-GULIJA, R. ZGORELEC & R. 
MAZURAN 2004: Application of short monolithic columns for fast purifi cation of plasmid DNA. 
Journal of Chromatography B 801 (2): 331–337.
BUNDESAMT FÜR GESUNDHEIT, (Eds) 2001: Schweizerisches Lebensmittelbuch.
CD-ROM, 311.510, BBL-EDMZ 3003, Bern, Switzerland 
CANKAR K., D. ŠTEBIH, T. DREO, J. ŽEL & K. GRUDEN 2006: Critical points of DNA quantifi cation by 
real-time PCR – effects of DNA extraction method and sample matrix on quantifi cation of gene-
tically modifi ed organisms. BMC Biotechnology 6: 37 – 51. 
GINZINGER D.G. 2002: Gene quantifi cation using real-time quantitative PCR: An emerging technology 
hits the mainstream. Experimental Hematology 30 (6): 503–512.
GRYSON N., K. MESSENS, K. DEWETTINCK 2004: Evaluation and optimisation od fi ve different extraction 
methods for soy DNA in chocolate and biscuits. Extraction of DNA as a fi rst step in GMO analysis. 
Journal of the Science of Food and Agriculture 84 (11): 1357–1363.
GRYSON N., F. RONNSE, K. MESSENS, M. DE LOOSE, T. VERLEYEN, K. DEWETTINCK 2002: Detection of 
DNA during the refi ning of soybean oil. Journal of the American Oil Chemists` Society 79 (2): 
171–174.
HUBER C. G. 1998: Micropellicular stationary phases for high-performance liquid chromatography of 
double-stranded DNA. Journal of Chromatography A 806 (1): 3–30.
JAMES C. 2006: Global status of commercialized biotech/GM crops: 2006. ISAAA Brief No. 35.  
JERMAN S., A. PODGORNIK, K. CANKAR, N. ČADEŽ, M. SKRT, J. ŽEL & P. RASPOR 2005: Detection of 
processed genetically modifi ed food using CIM monolithic columns for DNA isolation. Journal 
of Chromatography A 1065 (1): 107–113.
29Š. Höfferle, A. Kramar, F. Smrekar, D. Štebih, J. Vojvoda, J. Žel, P. Raspor: Anion-exchange …
HOLST-JENSEN A., M. DE LOOSE & G. VAN DEN EEDE 2006: Coherence between legal requirements and 
approaches for detection of genetically modifi ed organisms (GMOs) and their derived products. 
Journal of Agricultural and Food Chemistry 54 (8) :2799–809.
KARCHER S. 1995. Molecular biology: a project approach. San Diego, Academic Press, 280 pp.
LEVISON P. R., S. E. BADGER, P. HATHI, M. J. DAVIES, J. I. BRUCE & V. GRIMM 1998: New approaches 
to the isolation of DNA by ion-exchange chromatography. Journal of Chromatography A 827 (2): 
337–344.
LIPP M., A. BLUTH, F. EYQUEM, L. KRUSE, L. SCHIMMEL, G. VAN DEN EEDE & E. ANKLAM 2001: Validation 
of a method based on polymerase chain reaction for the detection of genetically modifi ed organisms 
in various processed foodstuffs. European Food Research and Technology 212 (4): 497–504.
LIPP M., P. BRODMANN, K. PIETSCH, J. PUWELS & E. ANKLAM 1999: IUPAC collaborative trial study 
of a method to detect genetically modifi ed beans and maize in dried powder. Journal of AOAC 
International 82 (4): 923–928.
MEYER R. 1999: Development and application of DNA analytical methods for the detection of GMOs 
in food. Food Control 10 (6): 391–399.
PODGORNIK A., M. BARUT, J. JANČAR, A. ŠTRANCAR & T. TENNIKOVA 1999: High-performance membrane 
chromatography of small molecules. Analytical Chemistry 71 (15): 2986–2991.
QUERCI M., M. MARETTI & M. MAZZARA 2004: Qualitative detection of MON810 maize, Bt-176 maize 
and Roundup Ready soybean® by PCR (Session 9). – In: QUERCI M., M. JERMINI & G. VAN DEN EEDE 
(eds.): Training course on – the analysis of food samples for the presence of genetically modifi ed 
organisms: user manual. Brussels, European Commission – Joint Research Centre, World Health 
Organization – Regional Offi ce for Europe, 30 pp. 
Regulation (EC) No. 1829/2003 of the European Parliament and of the Council of 22 September 
2003 on genetically modifi ed food and feed 2003. Offi cial Journal of the European Communities 
46 (L268): 1–23.
ROSSEN L., P. NORSKOV, K. HOLMSTROM, O. F. RASMUSEN 1992: Inhibition of PCR by components of 
food samples, microbial diagnostic assays and DNA-extraction solutions. International Journal 
of Food Microbiology 17 (1): 37–45.
SOMMA M. 2004: Extraction and purifi cation of DNA (Session 4). – In: QUERCI M., M. JERMINI, G. VAN 
DEN EEDE (eds.): Training course on – the analysis of food samples for the presence of genetically 
modifi ed organisms: user manual. Brussels, European Commission – Joint Research Centre, World 
Health Organization – Regional Offi ce for Europe, 18 pp. 
SZUHAJ B. F. 1983: Lecithin production and utilisation. Journal of  the American Oil Chemists` Society 
60 (2): 306–309.
ŠTRANCAR A., A. PODGORNIK, M. BARUT & R. NECINA 2002: Short monolithic columns as stationary pha-
ses for biochromatography. Advances in Biochemical Engineering/Biotechnology 76: 49–85.
TERRY C. F., N. HARRIS & H. C. PARKES 2002: Detection of genetically modifi ed crops and their de-
rivatives: critical steps in sample preparation and extraction. Journal of AOAC International 85 
(3): 768–774 
WURZ A., A. BLUTH, P. ZELTZ, C. PFEIFER & R.WILLMUND 1999: Quantitative analysis of genetically 
modifi ed organisms (GMO) in processed food by PCR-based methods. Food Control 10 (6): 
385–389.
ZIMMERMANN A., J. LÜTHY, U. PAULI 1998: Quantitative and qualitative evaluation of nine different 
extraction methods for nucleic acids on soya bean food samples. Zeitschrift für Lebensmittel-
Untersuchung und –Forschung A 207 (3): 81–90.