Anal Bioanal Chem (2010) 396:19771990
DOI 10.1007/s00216-009-3150-9
REVIEW
Influence of DNA extraction methods, PCR inhibitors
and quantification methods on real-time PCR assay
of biotechnology-derived traits
Tigst Demeke* & G. Ronald Jenkins
Received: 31 July 2009 / Revised: 4 September 2009 / Accepted: 9 September 2009 / Published online: 30 September 2009
# Canadian Grain Commission 2009
Abstract Biotechnology-derived varieties of canola, cotton,
corn and soybean are being grown in the USA, Canada and
other predominantly grain exporting countries. Although the
amount of farmland devoted to production of biotechnology-
derived crops continues to increase, lingering concerns that
unintended consequences may occur provide the EU and
most grain-importing countries with justification to regulate
these crops. Legislation in the EU requires traceability of
grains/oilseeds, food and feed products, and labelling, when
a threshold level of 0.9% w/w of genetically engineered trait
is demonstrated to be present in an analytical sample. The
GE content is routinely determined by quantitative PCR
(qPCR) and plant genomic DNA provides the template for
the initial steps in this process. A plethora of DNA extraction
methods exist for qPCR applications. Implementing stan-
dardized methods for detection of genetically engineered
traits is necessary to facilitate grain marketing. The Interna-
tional Organization for Standardization draft standard 21571
identifies detergent-based methods and commercially avail-
able kits that are widely used for DNA extraction, but also
indicates that adaptations may be necessary depending upon
the sample matrix. This review assesses advantages and
disadvantages of various commercially available DNA
*Contribution No. 1015 from the Grain Research Laboratory of the
Canadian Grain Commission.
T. Demeke (*)
Grain Research Laboratory, Canadian Grain Commission,
1404-303 Main Street,
Winnipeg, MB R3C 3G8, Canada
e-mail: tigst.demeke@grainscanada.gc.ca
G. R. Jenkins
United States Department of Agriculture, Grain Inspection,
Packers and Stockyards Administration,
10383 N. Ambassador Dr.,
Kansas City, MO 64153, USA
extraction kits, as well as modifications to published
cetyltrimethylammonium bromide methods. Inhibitors are a
major obstacle for efficient amplification in qPCR. The types
of PCR inhibitors and techniques to minimize inhibition are
discussed. Finally, accurate quantification of DNA for
applications in qPCR is not trivial. Many confounders
contribute to differences in analytical measurements when
a particular DNA quantification method is applied and
different methods do not always provide concordant results
on the same DNA sample. How these differences impact
measurement uncertainty in qPCR is considered.
Keywords DNA extraction . Sample matrices . DNA
quantification . PCR inhibitors . Variability of DNA yield
Introduction
A laudable goal for the future of the agricultural biotech-
nology industry is to identify efficient means of increasing
food and feed production, maintaining wholesomeness and
sustainability, without sacrificing quality or having a
detrimental effect on the environment. Many experts in
agricultural biotechnology deem genetically engineered
(GE) crop plants that confer insect resistance, herbicide
tolerance or combinations of these traits as a plausible
approach to meet such objectives. Second-generation
biotechnology crops will have genes for enhanced nutrition
and resistance to abiotic stresses, which will benefit
producers as well as consumers. Biotechnology-derived
crops have been grown in North America since the mid
1990s. In 2008, biotechnology-derived crops were grown in
25 countries on 125 million hectares of land [1]. The global
acreage of soybean, maize, cotton and canola continues to
increase, suggesting affirmation by government officials,
1978
research scientists, farmers and the agricultural industry of
the use and applications of this technology. However,
acceptance of GE crop plants into commercial markets has
not been without controversy. Although GE crops have
been widely adopted and used on a large scale in the USA,
Canada, Argentina and other grain-exporting countries,
acceptance of these crops has been more limited in Europe,
New Zealand and predominantly grain importing countries.
The EU implements regulatory regimes and legislative
mandates based on the process of GE crop production,
providing consumer choice through traceability and labelling
of GE-derived products (see [2] for EU genetically modified
organism regulations). Adventitious or a low-level presence
of GE grain in non-GE grain or food/feed products has also
been of international concern for countries that abide by the
Cartagena Protocol on Biosafety. In accordance with this
precautionary approach, living modified organisms must be
handled, transported, used and released in a manner that
minimizes risks to human health and biological diversity,
irrespective of overwhelming scientific data purporting
efficacy of these products (Article 18.2.a) [3]. Conversely,
Canada and the USA consider GE products to be substan-
tially equivalent when the nutritional content and a multitude
of safety aspects of the finished product are shown to be
indistinguishable from those of their non-GE counterparts
[4]. These fundamental differences in philosophies have led
to asynchronous regulations worldwide and a potential to
disrupt grain trade unless validated analytical methods are
implemented that (1) monitor the supply chain, (2) accurately
certify product content and (3) demonstrate compliance with
international labelling or traceability requirements [5].
One of the major challenges facing laboratories that test
for the presence of GE traits in food and feed products is
how to standardize testing procedures. To ensure compa-
rable analytical results by different laboratories, analyses
should be carried out with validated methods using
standard reference materials such that precision, accuracy,
sensitivity, specificity and robustness can be demonstrably
optimized. The most commonly used method to determine
GE content in food and feed products is the polymerase
chain reaction (PCR), although protein methods have
appropriate applications as well [6]. Accurate detection of
GE traits by PCR is dependent upon the specificity and
sensitivity of the amplification procedure as well as the
quality and quantity of genomic DNA that is dispensed into
the reaction.
The appropriate sampling method, sample size, type of
matrix, biological factors and inhibitors affect the quality
and quantity of DNA extracted from grain/seed, food and
feed samples. A universally accepted sampling method for
the detection and quantification of GE traits does not exist,
but helpful references on sampling methods are available
[79]. While ISO standard 21571 provides guidance
T. Demeke, G.R. Jenkins
annexes for extracting and quantifying genomic DNA with
publicly available methods, optimization conditions for
individual laboratory applications is commonly practised
[10]. Thus, a plethora of DNA extraction methods exist.
Once DNA has been extracted, prior to PCR, it must be
quantified. Development of accurate methods to quantify
DNA isolated from plant matrices is not trivial and several
methods for the quantification of DNA currently exist. All
DNA quantification techniques have limitations in their use
and application. This review considers the influence of
DNA extraction methods, quantification, and the presence
of PCR inhibitors that affect the performance and interpre-
tation of real-time PCR analytical results when testing for
the presence of biotechnology-derived traits in food and
feed products. This review does not cover real-time
quantitative PCR (qPCR) methods. Relevant references
for real-time qPCR methods are available [1113].
Commonly used DNA extraction methodstheir effects
on DNA quality and PCR
Real-time qPCR is a common method in agricultural
biotechnology for determining the GE content in food
and feed products. Trace amounts of biotechnology-
derived DNA are routinely analysed by qPCR to verify
the presence or absence of a particular trait, especially
during stages of product development, and for demon-
strating compliance with legislative mandates. Real-time
qPCR amplification is influenced by the quality, purity
and quantity of DNA in a sample. The aim of a nucleic
acid extraction method is to isolate DNA of suitable
integrity, purity and of sufficient quantity for diagnostic
applications by qPCR [14]. Obtaining DNA of high
quality is paramount for ensuring confidence in all
subsequent steps in the process of generating analytical
measurements. This section reviews DNA extraction
methods that are commonly utilized in agricultural biotech-
nology for diagnostic applications.
Cetyltrimethylammonium bromide method
The cetyltrimethylammonium bromide (CTAB) method has
been widely used for extraction of DNA from leaves, seeds/
grains and processed food/feed samples [10]. The procedure
is time-consuming and uses hazardous chemicals such as
phenol and chloroform. The basic CTAB extraction buffer
consists of 2% CTAB, 1.4 M NaCl, 20 mM EDTA and
100 mM tris(hydroxymethyl)aminomethane (Tris)HCl, pH
8.0. The original protocol [15] involved the use of CTAB
extraction buffer and chloroform/isoamyl alcohol (24:1) to
remove proteins and polysaccharides. Modifications to the
Influence of DNA extraction methods, PCR inhibitors and quantification methods on real-time PCR assay of biotechnology-derived traits 1979

CTAB protocol have involved addition of proteinase K and
mercaptoethanol to the CTAB buffer and the use of phenol/
chloroform/isoamyl alcohol (25:24:1) initially and then
cholorofrom/isoamyl alcohol (24:1) for further purification
[14, 16]. Addition of 10% N-phenacylthiazolium bromide
to the CTAB buffer has been reported to release DNA that
might be trapped within sugar-derived condensation prod-
ucts [17, 18]. There are several versions or modifications of
the CTAB protocol, as indicated in Table 1. It is important
to note that there are differences in the concentrations of the
CTAB extraction buffer components used for the different
versions. It is also worthwhile to determine the pros and

Table 1 Variations of cetyltrimethylammonium bromide (CTAB) DNA extraction methods

CTAB method Major steps involved in the procedure References

CTAB (original method) CTAB extraction buffer + chloroform/isoamyl alcohol + [15]

2-propanol ppt/70% ethanol wash + resuspend DNA in TE
CTAB + CsCl CTAB extraction buffer/mercaptoethanol + chloroform/isoamyl alcohol + 1/10th vol [68]
10% CTAB/0.7 M NaCl added to the supernatant + chloroform/isoamyl alcohol +
1% CTAB/50 mM Tris-HCl (pH 8.0)/10 mM EDTA + dissolve nucleic acid with
CsCl solution + dialysis to remove CsCl and partitioning to remove ethidium
bromide or use ethanol to precipitate DNA
CTAB + NaCl CTAB extraction buffer + choloroform + CTAB precipitation solution + resuspend [69]
DNA in 1.2 M NaCl + chloroform + 2-propanol ppt/70% ethanol wash +
resuspend DNA in sterile, deionized water. Used for qualitative PCR analysis
of GE materials in soybean and maize powder
CTAB/PTB CTAB extraction buffer/mercaptoethanol/PTB + chloroform/isoamyl alcohol + [17, 18]
2-propanol + dissolve pellet in homogenization buffer. Repeat the procedure
at least once. Used for food samples (soy flour, maize bread, cracker)/dried
and processed figs
CTAB + Genomic Tip 20 CTAB extraction buffer/RNase A/proteinase K + chloroform (2) + CTAB [19]
precipitation buffer + DNA resuspended in 1.2 M NaCl + chloroform + absolute
ethanol for precipitation + 70% ethanol wash + nuclease-free H2O to resuspend DNA +
Genomic Tip 20 column for further purification. Used for maize flour material
CTAB + SDS Extraction buffer/RNase A/SDS + potassium acetate + 2-propanol precipitation/70% ethanol [70]
wash + resuspend DNA in TE/H2O + clean-up solution (has CTAB) + dissolve
DNA in H2O + use sodium acetate and ethanol to precipitate DNA + 70% ethanol
wash + dissolve DNA pellet in TE buffer. No phenol or chloroform was used. Not
tested for detection of GE samples
CTAB (ISO) CTAB extraction buffer/-amylase solution/RNase A/proteinase K + chloroform + Annex A3 in [10]
CTAB precipitation buffer + DNA resuspended in 1.2 M NaCl + chloroform +
2-propanol ppt/70% ethanol wash + resuspended DNA in H2O or TE. Method
used to extract DNA from cereal grains and food matrices
CTAB/chloroform + CTAB extraction buffer/RNase A/proteinase K + chloroform (2) + CTAB [42]
NaCl/chloroform precipitation buffer + DNA resuspended in 0.5 TE + RNase A treatment + add
equal volume of 2.4 M NaCl + chloroform + absolute ethanol for precipitation + 70%
ethanol wash + DNA dissolved in 0.5 TE. Used for maize flour
CTAB + Zymo kit or CTAB + CTAB extraction buffer/proteinase K/mercaptoethanol + phenol/chloroform/isoamyl [16]
Qtip 100 alcohol (1) + chloroform/isoamyl alcohol (2) + 2-propanol precipitation/70% ethanol
wash + RNase A and RNase T1 treatment + resuspend DNA in TE + kit
purification (Zymo or Qtip 100). Used for extraction of DNA from soybean flour
CTAB + Wizard resin + CTAB extraction buffer/proteinase K/RNase A + chloroform + CTAB precipitation [20]
microSpin column solution/1.2 M NaCl + chloroform + 2-propanol precipitation + purification with
Wizard DNA clean-up system + purification using S-300 HR microSpin columns.
Used for soybean seeds and ground maize grain/seed
CTAB + PEG CTAB extraction buffer/mercaptoethanol/proteinase K + phenol/chloroform/isoamyl [71]
alcohol (3) + 2-propanol precipitation + TE/RNase treatment + chloroform/isoamyl
alcohol (3) + precipitate DNA with ammonium acetate and ethanol + TE/PEG
precipitation + resuspend in TE or water. Used for canola seeds
CTAB + micro and ultra CTAB extraction buffer (has PVP) + chloroform/isoamyl alcohol (1) + RNase [21]
filtration A treatment of the supernatant + chloroform/isoamyl alcohol (1) + resuspend
in TE + micro and ultra filtrations (Nanosep MF 0.2 + Pall Nanosep 30 K).
Used for maize seeds

It is important to note that there are variations in the concentrations of the CTAB extraction buffer components used among some of the methods.
N-Phenacylthiazolium bromide (PTB) (10% solution) cleaves macromolecule-derived protein cross-links [17].
TE tris(hydroxymethyl)aminomethane/EDTA, Tris tris(hydroxymethyl)aminomethane, GE genetically engineered, SDS sodium dodecyl sulphate,
PVP polyvinylpyrrolidone
1980
cons before deciding on the type of CTAB extraction
method to use. For real-time PCR use, RNA is removed
from the nucleic acid preparation with enzymes such as
RNase A and RNase T1.
According to published reports, CTAB-extracted
DNA needs further purification to be used for real-
time PCR [16, 1921]. Purification of CTAB extracted
DNA with a Genomic Tip 20 column (Qiagen) resulted in
a linear calibration curve and produced the expected
values [19]. The A260/A230 ratio of CTAB-extracted DNA
was low (average of 1.29), indicative of contamination
with compounds such as polysaccharides which may
inhibit qPCR [16]. Dilution of the CTAB-extracted DNA
(25 ng total DNA instead of 100 ng total DNA in the
25-L PCR) resulted in reasonable quantification of
Roundup Ready soybean (GTS 40-3-2). Further purifi-
cation of the soybean DNA with either the Zymo kit
(Zymo Research) or Qtip 100 (Qiagen) provided clean
DNA that was suitable for real-time PCR [16]. Purifica-
tion of CTAB-extracted canola DNA with the Zymo kit
also produced clean DNA that was suitable for real-time
PCR (T. Demeke and I. Ratnayaka, personal observation).
Some of the modified CTAB DNA extraction methods
may be more effective in removing inhibitors than the
others. CTAB-based DNA extraction methods have been
validated by the Community Reference Laboratory for GM
Food and Feed [20, 21].
Sodium dodecyl sulphate and polyvinylpyrrolidone
methods
The basic sodium dodecyl sulphate (SDS) extraction buffer
consists of 100 mM Tris-HCl pH 8.0, 50 mM EDTA pH
8.0, 500 mM NaCl, 10 mM mercaptoethanol and 1% SDS
[22]. The concentrations of the extraction buffer compo-
nents may vary. Proteins and polysaccharides are removed
as a complex with the insoluble potassium dodecyl
sulphate. SDS has also been used in combination with
phenol/chloroform (see Annex A.1 in [10]) to successfully
extract DNA from various matrices. The SDS-based DNA
extraction method in combination with phenol/chloroform
has also been validated by the Community Reference
Laboratory for GM Food and Feed [23].
The polyvinylpyrrolidone (PVP) method is suitable for
matrices containing a high amount of polyphenolic com-
pounds [10, 24]. The method includes thermal lysis in the
presence of SDS and EDTA followed by addition of PVP
and ammonium acetate to remove contaminants such as
polyphenolic compounds and polysaccharides. The ISO
21571 method (Appendix A.2) has been used to extract
DNA from various matrices such as corn flakes, tofu and
cereal bars [10].
T. Demeke, G.R. Jenkins
DNA extraction with commercially available kits
Most commercially available kits and published protocols
utilize detergent, to disrupt the cell wall, as an initial step for
extracting DNA from plant material. Second, to eliminate
contaminating RNA and proteins, RNase A treatment and
proteinase K digestion is routinely performed. Some commer-
cial kits utilize DNA binding to silica-based matrices or
magnetic beads, followed by elution, to avoid exposure to
organic solvents such as chloroform. Other methods selectively
isolate DNA from cellular debris using high salt concentrations
and ethanol precipitation, followed by subsequent ethanol
washes. Examples of commercially available DNA extraction
kits are listed in Table 2 and a summary of different
approaches that are used for DNA extraction is presented in
Table 3. Many of the commercially available kits can be used
for both DNA extraction and DNA purification. A limited
sample size (e.g. 20200 mg) is generally used for DNA
extraction with kits. DNA extraction kits are generally faster
than CTAB- and SDS-based methods; however, they may be
more expensive. The cost of DNA extraction per sample for a
given method is not often reported in the literature. For potato
DNA extraction, the cost per sample (Canadian dollars) was
reported to be $0.50 for CTAB, $2.15 for Wizard resin
(Promega), $2.50 for Hi-Pure (Roche), $3.20 for UltraClean
plant DNA kit (Mo-Bio), $3.70 for Wizard magnetic DNA
purification system for food (Promega) and DNeasy plant
mini kit (Qiagen) and $5.90 for DNA isolation kit for cells
and tissues (Roche I) [25]. Labour and instrument costs were
not included in the analysis.
Kits such as DNeasy and Wizard have been widely used for
DNA extraction from various matrices for GE analysis by
PCR. The success of the DNA extraction depends upon the
kind of plant material and matrix used. A kit found to be
suitable for DNA extraction of one kind of matrix may not be
suitable for a different kind of matrix. For example, the Wizard
DNA extraction kit was found to be suitable for corn flour and
corn starch [26] and potato derived food stuffs [25]. On the
other hand, the Wizard kit was not suitable for soybean and
maize grains and processed food/feed stuffs [27].
For seeds and flours of maize and soybean, QIAamp DNA
stool minikit produced good-quality DNA with a low level of
degradation. Real-time quantification of Roundup Ready
soybean content with DNA extracted by the QIAamp DNA
stool kit resulted in the highest correlation with the expected
values[ 28]. The High Pure GE sample preparation kit (Roche)
has been reported to be the most reliable DNA extraction
method in comparison with CTAB/N-phenacylthiazolium
bromide, QIAamp DNA stool kit, Master Pure DNA
purification kit and NucleoSpin food kit for food samples
[18]. For complex foodstuffs such as crackers, tacos and tofu,
NucleoSpin food kit (MachereyNagel) produced relatively
high quality DNA with a low level of degradation [28].
Table 2 Partial list of commonly used plant DNA extraction methods and kits

Extraction method Description Examples of plants References

CTAB lysis and purificationvariations of Effective for a wide range of matrices. Complexes out A wide range of plants Annex A3 in [10]
CTAB DNA extraction methods are polysaccharides and proteins. Used with phenol/chloroform: and matrices and [14, 15]
provided in Table 1 isoamyl alcohol (hazardous). Takes a long time
DNA Clean & Concentrator 25 kit Uses fast spin column technology. Used for DNA purification Soybean, canola [16]
(Zymo Research)
DNeasy (mini and maxi-kits) (Qiagen) Uses silica-gel-membrane and simple spin procedures to isolate DNA. Non-toxic Corn, soybean [19, 27, 31]
Epicentre MasterPure Complete Utilizes rapid salt precipitation protocol to remove contaminating macromolecules Soybean, maize [18]
(EPICENTRE Biotechnologies)
Fast ID genomic DNA extraction kit/Fast ID Uses genomic lyse and bind buffers as well as DNA binding columns Soybean, maize, rice, [72]
food DNA extraction kit (Genetic ID) processed food and
feed samples
GenElute plant genomic DNA Uses silica binding and elution in a spin column format Maize flour [73]
miniprep kit (Sigma)
GENESpin (GeneScan) Based on silica membrane spin column technology Soybean, maize, canola, [27]
processed food, cereals,
flour and lecithin
Genomic-Tip 100/G (Qiagen) Uses anion-exchange technology. Used for DNA purification Soybean [16]
GM quicker (Nippon Gene; bioMerieux) Uses silica spin column and anionic detergent Soybean, maize [19, 74]
High Pure GE sample preparation kit (Roche) DNA is bound selectively to special prepacked glass fibres and a Soybean, maize [18]
low salt elution releases the DNA from the glass fibre
Nanosep and Nanosep MF centrifugal Ultrafiltration system Maize seeds [21]
devices (Pall Corporation)
NucleoSpin food kit (MachereyNagel), Uses silica membrane spin technology to bind to the DNA. Brassica, maize, soybean, [18, 75]
Clontech products Allows processing of 50200-mg sample feed, processed food
PVP method Includes thermal lysis in the presence of SDS and high EDTA concentration Used for various matrices Annex A2 in [10]
followed by removal of contaminants such as polyphenolic compounds and
polysaccharides. Suitable for matrices containing a high amount of
polyphenolic compounds
QIAamp DNA stool kit (Qiagen) Silica-membrane-based purification system. Suitable for PCR-inhibitor-rich Soybean, maize [28, 76]
substances and for highly processed foodstuffs
QIAEX II gel extraction kit (Qiagen) Based on selective adsorption of nucleic acids onto QIAEX II silica-gel Soybean, canola [14, 35]
particles in the presence of chaotropic salt. Used for DNA purification. Lipase
treatment may be required for fat-rich matrices
SDS Improves lysis of cells. Widely used for DNA extraction from seeds. SDS is also Used for a wide range [10, 22]
used in combination with phenol/chloroform of plants
UltraClean plant DNA kit (Mo-Bio) Cell lysis achieved with bead grinding; silica binding; spin column format. Potato [25]
Quantifiable DNA was not recovered from potato tubers.
Wizard magnetic DNA purification Uses magnetic separation with MagneSil PMPs Corn, soybean [19, 27]
system (Promega)

PMPs paramagnetic particles

Influence of DNA extraction methods, PCR inhibitors and quantification methods on real-time PCR assay of biotechnology-derived traits 1981
1982
Table 3 Summary of different approaches used for DNA extraction
DNA extraction Cell Protein Polysaccharide
method lysis precipitation precipitation
Qiagen
DNAzola
Wizard
NucleoSpin
Gentrab
CTAB
a
DNAzol (Molecular Research Centre) extraction is based on the use of a novel guanidine-detergent solution that hydrolyses RNA and allows the
selective precipitation of DNA.
b
Gentra Puregene blood kits (Qiagen) allow purification of high molecular weight DNA.
Effects of sample matrices on DNA quality and PCR
The quality and quantity of DNA extracted from food products
tend to decrease with the extent to which the food is processed
[25, 28]. Exposure to heat results in fragmentation of
high molecular weight DNA, and physical and chemical
treatments cause random breaks in DNA strands, reducing the
DNA fragment size [18]. The type of sample used for DNA
extraction will influence the quality and quantity of DNA.
Intact and high-quality DNA can be obtained from ground
samples of grains and oilseeds. Conversely, DNA extracted
from highly processed food and feed samples is generally
more sheared and of low molecular weight. For highly
sheared DNA, there may not be enough templates available
for PCR and this will have an impact on the limit of detection
and quantification. Only minute amounts of DNA can be
extracted from refined oil, modified starch, etc. and this will
affect the reproducibility and quantification of DNA by PCR.
Complex sample matrices may require multiple consecutive
DNA extraction and purification steps [12]. A DNA extraction
method used for grain samples may not be suitable for
processed samples. Studies have been conducted to determine
whether the percentage of GE product obtained from a
processed food reflected the original percentage of GE
product in the raw material [29, 30]. These studies demon-
strate that in some instances processed foods provide DNA
material that is not suitable for measuring GE content by
qPCR since the copy number of the trait or both the trait- and
taxon-specific genes were below the quantification limit of 20
copies of PCR-amplifiable DNA. In other processed food
products, where copy number was not limiting, high bias and
a statistically significant difference was observed in the
measured GE content of processed foods (containing mostly
degraded DNA) compared with their raw counterparts
(consisting of mostly intact DNA) [30].
DNA extraction efficiency is unequivocally dependent
upon particle size. As the particle size in a sample decreases,
yields increase [31, 32]. When DNA yields amongst corn
T. Demeke, G.R. Jenkins
Silica Organic Magnetic DNA DNA
gel solvent beads precipitation elution
samples of various particle sizes (i.e. flour, meal and grits)
were compared, corn flour with the finest particle size
provided the highest yield [33]. This observation can be
easily explained by the fact that smaller particles offer a
larger surface area of exposure to extraction reagents
compared with larger particles. Relative GE content quanti-
fication could be significantly affected if the analysed food
contains fractions with different particle size distributions.
DNA extractions from fine (4463 M) and coarse (102
115 M) corn powders did not, however, influence the DNA
copy number estimation [34]. The particle sizes of GE and
non-GE ground samples need to be similar for appropriate
comparison with real-time qPCR. Routine grinding to obtain
fine particle sizes can be cumbersome and time-consuming,
especially for oil-rich seeds such as canola and soybean.
DNA extracted from samples of ground canola and soybeans
that passed through a 1.0-mm sieve have been used
successfully in real-time qPCR assays [16, 35].
Biological challenges and variability of DNA yield
among cultivars, plants and processed foods
Analysis of GE content by qPCR in plant sources involves
measurement of copy numbers of a trait-specific target gene
and a taxon-specific, endogenous reference gene. Analysis of
the GE content in plant material by qPCR faces several
biological challenges, all of which contribute to measurement
uncertainty. For example, when the levels of GE content are
quantified by qPCR, the ratio between the target gene and the
endogenous reference gene is assumed to be the same for any
given test or reference sample. However, this assumption is
not valid under certain circumstances [12, 36]. The Institute
for Reference Materials and Measurements (IRMM) maize
event Bt-11 contains two copies of the 35S promoter per
copy of maize endogenous reference gene [12]. Another
biological confounder that contributes to measurement
uncertainty with qPCR is the genetic composition of plant
Influence of DNA extraction methods, PCR inhibitors and quantification methods on real-time PCR assay of biotechnology-derived traits 1983
cultivars and genome size. Zea mays contains between 2,292
million and 2,716 million base pairs per haploid genome,
suggesting that different plant cultivars contribute different
copy numbers when equivalent mass amounts are dispensed
into the qPCR [37]. Reports also suggest that DNA yields
may not be the same amongst different cultivars, with some
yielding up to 20% less DNA when a CTAB extraction
method is used [32, 38]. Ten maize cultivars had ratios of
endosperm to total DNA ranging from 36.3 to 59.4%,
compared with ratios of embryo to total DNA which ranged
from 38.6 to 59.6% [32]. However, variation in the
endosperm DNA did not significantly affect the quantifica-
tion of GE content in this study. Characterization studies
suggest that the maize kernel is made up of 83% endosperm,
11% embryo, 5% pericarp and 1% tip cap [39]. In a single
kernel analysis, endosperm plus pericarp contained approx-
imately 10 times more extractable DNA than did embryo
[39]. Other reports suggest that about half of the total DNA
extracted from maize kernels originates from endosperm and
the other half from embryo [32]. These findings raise
concerns about accurate quantification of GE content using
qPCR considering that the endosperm consists of two
maternal haploid genomes and one paternal haploid genome,
whereas the embryo consists of one each of a maternal and a
paternal haploid genome. DNA endoreduplication (duplica-
tion of the genome without mitosis) in maize also poses a
challenge for qPCR. Endoreduplication leads to polyploid-
ization, in which a single cell contains several copies of the
genome [12], and may impact the relative contribution of
embryo and endosperm in ground maize samples. Since
quantification by qPCR cannot distinguish endoreduplication
or zygosity differences between test and reference samples,
the genetic make-up of a test sample and its reference must
be the same with respect to zygosity, ploidy and endoredu-
plication status, lest an increase in measurement uncertainty
(i.e. maize kernels homozygous for a given event contribute
more copies of the target gene compared with maize kernels
containing the same event in heterozygous state). In the
context of transgene identification and quantification, endor-
eduplication amplifies the whole genome and does not alter
the ratio of the copy numbers [36].
Sufficiently high yields of intact genomic DNA can usually
be obtained from grains, oilseeds or minimally processed food
samples. Conversely, it becomes more challenging to obtain
high yields of intact genomic DNA from food and feed
products that have been subjected to mechanical, thermal,
chemical and enzymatic treatments [14, 40]. When structural
damage to DNA occurs, the process appears to be entirely
random [41]. Various models have been proposed to describe
the length of a target DNA sequence and its probability of
being damaged by random scission [41]. It is common to
design primers/probe of relatively short length to amplify
short segments of the target sequence. Many highly
processed food and feed products contain genomic DNA
from a multitude of plant and animal sources. Under these
conditions, when a specified quantity of DNA is added into
the PCR, only a small proportion of molecules will contain
the target of interest with appropriate length for amplifica-
tion. To improve the reliability of PCR, normalizing to a
taxon-specific reference (endogenous control) gene becomes
critical [42].
Several studies evaluating DNA extraction methods to
characterize yield, quality and predictability in PCR can be
found in the literature. Identifying the most appropriate
method for a particular type of food product, one that
provides DNA of the highest quality and sufficient yield for
analysis of a particular food product, is a daunting task [28,
30, 40, 43]. Most studies have been designed to characterize
a representative food type with different extraction methods.
For example, out of nine different extraction methods
compared for soybean food samples, five methods provided
predominantly degraded DNA, whereas the other four
methods provided relatively intact DNA but with signifi-
cantly lower yields [43].
The complexity of detecting GE traits in grains
continues to grow as new varieties are introduced into
the marketplace [44]. The future trend for the agricultural
biotechnology industry is to develop cultivars with
stacked GE traits (e.g. maize). Only single kernel (or
plant) analyses can differentiate between multiple events
stacked into a single cultivar and a mixture of events
derived from a mixture of cultivars [45, 46]. However,
single kernel analysis using PCR to detect the presence of
stacked events requires testing of a large number of seeds
per lot and is not economically feasible with currently
available PCR technologies.
Overall, use of certified reference materials, validated
DNA extraction and PCR methods will help to overcome
some of the problems discussed above. A guideline on
identification and quantification of stacked GE events will
be useful in the near future.
Types of PCR inhibitors and methods to reduce
their impact
Inefficient PCR amplification may result if an insufficient
quantity of DNA of appropriate molecular weight is used or
may be due to the presence of inhibitors in the DNA
extract. Inhibitors in the DNA can reduce the efficiency
and/or reproducibility of PCR and thus may contribute to
inaccurate qPCR results. The inhibitory mode of action of
some of the compounds may be linked with precipitation of
the DNA, denaturation of DNA or the ability of the
polymerase enzyme to bind to magnesium ions [47].
Inhibitors may kinetically modify PCR amplification by
1984
chelating Mg2+, a cofactor for all DNA polymerases,
including Taq polymerase, or by binding to target DNA or
the DNA polymerase [48]. Enzyme inhibitors may originate
from either the plant tissue or the reagents used for DNA
isolation [49].
The major types of PCR inhibitors are listed in Table 4.
Acidic plant polysaccharides (e.g. dextran sulphate, alginic
acid) have been reported to be inhibitory, whereas neutral
polysaccharides (e.g. starch, arabinogalactan) were not
found to be inhibitory [31, 49, 50]. The presence of
0.001% or more dextran sulphate resulted in complete
inhibition of PCR [31]. On the other hand, the presence of
4% dextran (a neutral polysaccharide) did not have an
effect on PCR. DNA solutions can become viscous owing
to the presence of polysaccharides and other substances. It
is difficult to accurately pipette a solution of highly viscous
DNA. Centrifugation of soybean DNA solution [resuspended
in Tris-HCl/EDTA (TE)] at low speed (e.g. 7,000 rpm) for
5.0 min resulted in pelleting out of the viscous material [16].
The supernatant can be used for qualitative PCR, even
though further purification with a kit may be required for
real-time PCR (Table 2).
Process-introduced PCR inhibitors include phenol,
EDTA, sodium chloride and lithium chloride. Phenol at a
concentration of more than 0.2% inhibits PCR [49, 51].
Phenolic compounds from the sample or carried over from
DNA purification can inhibit PCR by binding to or
denaturing the polymerase [48]. Generally, plant phenolic
compounds (e.g. chlorogenic acid, caffeic acid) can react
Table 4 Examples of PCR inhibitors reported in the literature and methods to minimize inhibition
Inhibitors Description and inhibitory concentration for PCR
CTAB 0.005% (w/v) [49, 51]; 0.01% [47]
EDTA 0.5 mM [49, 51]; 1 mM [47]
Ethanol >1% (v/v) [49]
Fat
Isopropanol >1% (v/v) [49]
Phenol >2% (v/v, [49]; >0.2% [51]
Chlorogenic acidplant phenol (0.240.36 g/L)inhibited
PCR in potato [53]
Polysaccharides Acidic polysaccharides such as dextran sulphate
are inhibitory [14, 50]
Dextran sulphate: >0.1% [51]; 0.001% [30]
Pectin: >0.5% [49]
Xylan: >0.0025% [51]
Protein 1% casein hydrolysate in PCR mixture caused inhibition [47].
SDS 0.005% (w/v, [49]
Sodium acetate 5 mM [49]
Sodium chloride 25 mM [49]
T. Demeke, G.R. Jenkins
with proteins/enzymes and alter various properties of the
biopolymers [52]. In potato, a chlorogenic acid concentra-
tion of 0.240.36 g/L inhibited reverse transcriptase
PCR [53]. An EDTA concentration of 0.5 mM or higher
has been reported to inhibit PCR (Table 4). At the end of
the extraction process, DNA is usually resuspended in 1
TE (10 mM Tris-HCl and 1 mM EDTA). The EDTA may
be reduced (e.g. 0.1 or 0.5 TE) or eliminated completely.
Only 10 mM Tris-HCl, pH 8.0 (without EDTA) is also
being routinely used to resuspend DNA that is used for
real-time PCR. DNA can also be resuspended in distilled
sterile water; however, in the absence of stabilizing
compounds the DNA cannot be stored for a long period
of time [54].
Concentrated protein (e.g. 1% casein hydrolysate) has
been reported to inhibit PCR [14, 47]. Ionic detergents such
as SDS are reported to be more inhibitory than non-ionic
detergents such as Tween 20 and Triton X-100 [47]. CTAB
and SDS concentrations of more than 0.005% inhibited
PCR [49]. The two detergents are usually used early during
DNA extraction, and thus residual amounts are removed
through the subsequent washing steps. Ethanol and 2-
propanol are routinely used for DNA extraction even
though they could inhibit PCR. 2-Propanol and ethanol
are reported to inhibit PCR at concentrations of 1% or more
[49]. Drying the DNA pellet prior to resuspension removes
residual ethanol and 2-propanol.
Powder materials from gloves worn in the laboratory
have been shown to inhibit PCR. Washing gloves or using
Methods to minimize inhibition
70% ethanol wash
Reduce the concentration of EDTA to 0.1 mM in the TE
buffer or simply use Tris-HCl (10 mM) to bring DNA in
solution. DNA can also be brought in pure water (but the
DNA cannot be stored for long-term use)
Dry pellet and resuspend
Lipase or hexane treatment and chloroform extraction [14]
Dry pellet and resuspend [14]
Use PVP, PVP/ammonium acetate [14]
Incorporation of 1.2% citric acid at the DNA extraction step
neutralized the inhibitory effect of chlorogenic acid
Use CTAB buffer and chloroform extraction. Treatment with
enzymes such as pectinase, cellulase, hemicellulase and
-amylase can be used to remove polysaccharides High
salt precipitation
Use SDS, CTAB or guanidinium buffers, proteinase K
Wash with 70% ethanol
Wash with 70% ethanol
Wash with 70% ethanol or use silica-based purification [14]
Influence of DNA extraction methods, PCR inhibitors and quantification methods on real-time PCR assay of biotechnology-derived traits 1985
non-powdered types will help to minimize the inhibition
[48]. Commercially available kits are being widely used for
purification of DNA extracted by CTAB and other methods
(Table 2). Some of the kits are used for both extraction and
purification of DNA. Dilution of the DNA helps to reduce
the inhibitor concentration and enhance PCR efficiency;
however, a lower DNA concentration may decrease PCR
sensitivity. An inhibition test using either internal controls
or evaluation of the linearity of the calibration curves
should be performed to determine the suitability of the
extracted DNA for real-time PCR amplification [12, 19].
DNA quantification technologies
Prior to qPCR, stock DNA extracts are commonly
quantified and diluted so that all reference and test samples
contain identical amounts of DNA. Accurate determination
of DNA concentration in a sample is a critical component
for analysis of GE traits by qPCR. It may be of little value
to have a method for measuring DNA concentrations with a
high level of accuracy if the extracts themselves do not
yield pure DNA. In general, DNA quantification prior to
qPCR increases confidence in negative PCR results in
diagnostics where insufficient target DNA could otherwise
be interpreted as a false-negative result. The most com-
monly used techniques for DNA quantification include UV
spectrophotometry, fluorometry, chemiluminesence and
agarose gel electrophoresis. These are discussed in more
detail in the following sections.
UV spectrophotometry
Concentrations of nucleic acid solutions, for PCR and other
molecular biology applications, are commonly quantified
by measuring the absorbance at 260 nm (A260). For DNA
that is predominantly intact, a solution with an A260 value
that equals one optical density (OD) unit corresponds to a
sample that contains 50 g DNA/mL. For DNA that is
largely degraded or consists of single-stranded DNA
(ssDNA), an A260 value of 1 OD unit was found to
correspond to 38 g DNA/mL [55]. For proteins in
solution, an A280 value that equals 1 OD unit corresponds
to a protein level of 1 g/mL [56]. Protein contamination of
DNA is routinely assessed by comparing the A260/A280
ratio; with a value of 1.7 or greater being acceptable [57].
Contaminants including RNA, single-stranded nucleic
acids and phenols contribute to the total signal in an A260
measurement and are not discernible from double-stranded
DNA (dsDNA). The degree to which DNA degradation
occurs and the ratio of ssDNA to dsDNA may vary from
extract to extract, suggesting uncertainty in any DNA
quantification measurement by UV spectrophotometry. As
dsDNA becomes more highly denatured, the A260 value
routinely increases accordingly. The observed A260 increase
in degraded DNA samples is reportedly due to hyperchromic
effects wherein strand separation and base pair unstacking
contribute to an increase in the absorbance signal above that
of purely double stranded DNA [58, 59]. When DNA is
denatured, interactions between base pairs can become
disrupted as the double-helix structure of DNA dissociates
into single strands. As a result, the bases assume a larger
number of resonance structures and thus A260 increases.
Upon purification, DNA solutions might not always be
homogeneous. In these instances, it is often difficult to measure
DNA concentrations precisely and accurately. Inhomogeneity
may occur when DNA is obtained in a highly concentrated
form, predominantly of high molecular weight [57]. Quanti-
fication by UV spectrophotometry generally requires rela-
tively large amounts of DNA, typically 5.050 g DNA/mL
per sample A260 0:1
1:0 OD units, compared with
fluorescent-dye methods, which typically require 0.03
0.25 g DNA/mL [57, 60]. In summary, accuracy and
precision of DNA quantification by UV spectrophotometry
can be influenced by (1) the size distribution of DNA in
solution, (2) the ratio of ssDNA and dsDNA and (3) the
presence of other components that absorb at 260 nm, such as
RNA and single nucleotides.
Fluorometry
Fluorometry is a useful, rapid, relatively inexpensive method
for quantifying dsDNA prior to qPCR. For measurement of
the concentration of DNA, fluorometry has a sensitivity
similar to that of chemiluminescence (as described later) and
both techniques are more sensitive than spectrophotometry.
Preparations of serially diluted standards and test samples are
incubated with a fluorescent dye such as Hoechst 33258 or
PicoGreen reagent. Fluorescence values of the standards are
used to construct a linear regression against which the
concentrations of the test samples are estimated by interpola-
tion. The linear concentration range for DNA quantification
extends over 4 orders or magnitude (25 pg/mL to 1 g/mL)
using a single dye concentration. Assay linearity is maintained
in the presence of proteins, urea, ethanol and chloroform but
not ionic detergents.
Factors that influence accuracy and precision of DNA
quantification using fluorometry include (1) the size distribu-
tion of DNA in solution, (2) exclusivity of dyes (i.e. some
dyes are more specific for dsDNA), (3) sensitivity of different
dyes and their optimal working concentration ranges, (4)
DNA integrity, (5) sensitivity of the assay to photobleaching
and (6) DNA purity [61]. Hoechst 33258 requires high salt
concentrations to detect dsDNA in the presence of RNA and
1986
low salt concentrations to detect dsDNA in the presence of
ssDNA, suggesting that two different assay solutions are
required to obtain selectivity when samples that contain both
RNA and ssDNA are analysed. Also, Hoechst 33258 is AT-
selective, whereas PicoGreen reagent is not base-selective.
PicoGreen tends to show greater dsDNA:RNA selectivity
compared with Hoechst 33258 [61]. The PicoGreen assay
is suitable for quantifying dsDNA prior to PCR and for
quantifying post-PCR products but is not suitable for thermal
cycling as when melting steps are included. PicoGreen dye
is sensitive to salts, especially divalent cations and ionic
detergents. Some reports suggest as there is as much as
94.6% decrease in signal intensity with PicoGreen when
genomic DNA contains 0.005% CTAB detergent [60].
Chemiluminescence
Chemiluminescence is a highly sensitive method, designed
for the specific detection of linear, dsDNA [62]. When this
method of DNA quantification is used, chromosomal plant
DNA must be reduced to fewer than 6,000 base pairs in
length by an appropriate restriction enzyme digestion prior
to the assay being performed [63]. When high molecular
weight genomic DNA is used in this assay, the light output
does not produce the same signal as an equivalent mass of
smaller fragmented DNA. Restriction enzymes producing
blunt-ended, 5 overhang or 3 overhang DNA provide
results concordant with A260 measurements [63]. The
measurement is based upon two coupled enzymatic
reactions that produce a light signal proportional to the
amount of linear dsDNA in the sample (see Fig. 1) [63].
DNA is depolymerized, the deoxynucleotides are converted
to ATP and luciferase enzyme is added, which allows for
ATP quantification. The amount of ATP is measured by a
luciferase/luciferin reagent. The light signal produced in the
reaction is proportional to the original amount of linear
dsDNA present in the sample. The absolute amount of
DNA in a test sample is generally compared with the light
output measured from standards. The linear range of this
assay is between 20 and 1,000 pg of total DNA per
reaction. The amount of ATP generated peaks after
approximately 10 min. The enzyme ATPase can signifi-
cantly affect the accuracy of this assay system. Quantifica-
Fig. 1 Quantification of DNA using a chemiluminescence system. dsDNA double-stranded DNA. (Reproduced with permission from Promega)
T. Demeke, G.R. Jenkins
tion by chemiluminescence is less affected by contaminants
than spectrophotometry and fluorometry.
Agarose gel electrophoresis
Agarose gel electrophoresis is a commonly used method to
separate DNA and RNA molecules of different sizes but can
also be used to assess DNA quality and provides useful
information regarding the amount of DNA in a solution. Since
DNA has a consistent charge-to-mass ratio, the major factor
that influences its migration through a gel matrix is size [57].
Smaller DNA molecules migrate through gels at a faster rate
compared with DNA of higher molecular weight. By using
gels with different percent concentrations of agarose, one can
resolve different sizes of DNA fragments. Higher concen-
trations of agarose facilitate separation of smaller DNA
molecules, whereas lower agarose concentrations allow
resolution of larger DNA molecules. Agarose concentrations
of 0.81% are typically used for quantification of genomic
DNA. Once electrophoresis is complete, the DNA in the gel
is visualized by staining with a fluorescent dye (e.g. ethidium
bromide, GelRed, SYBR green), followed by illumination
with UV light. Ethidium bromide intercalates between bases
of nucleic acids and allows very convenient detection of
DNA fragments in gels. The fluorescence of the dyeDNA
complex is greater than that of the unbound dye. The
presence of an intense, high molecular weight band in the gel
indicates intact genomic DNA with minimal degradation.
Degraded or sheared DNA and RNA contamination can be
visualized as a smear towards the lower molecular weight
portion of the gel [42]. DNA concentrations can be estimated
semiquantitatively by running serially diluted DNA, of
known concentration, and visually comparing the intensity
of the fragment bands with the intensity of the bands of test
samples of unknown concentrations.
Limitations of DNA quantification technologies
and effects of degraded compared with intact DNA
on qPCR
The purpose of an extraction method is to obtain highly
pure DNA that provides an acceptable quantity of material
Influence of DNA extraction methods, PCR inhibitors and quantification methods on real-time PCR assay of biotechnology-derived traits 1987
that is highly intact with minimal degradation. Once DNA
has been extracted, the level of DNA degradation that
occurs varies significantly and is contingent upon the
sample source, processing, extraction protocol and other
contributing factors. Also, the degree of degradation
between reference and test sample DNA can vary signifi-
cantly. Reports suggest that when DNA is highly degraded,
and the targets for the GE and reference genes differ
significantly in size, the reference assay may not normalize
the GE content correctly [64]. Agarose gel electrophoresis
enables visualization, to some extent, of the degree of DNA
degradation within a specified sample. Generally, levels of
DNA degradation are more pronounced in processed foods
and feeds as compared with IRMM reference material [64,
65]. In theory, the final qPCR measurement assigned to a
particular sample should be independent of the degree of
DNA degradation since GE target sequence and taxon-
specific sequences degrade congruently. In practice, some
reports suggest that variability in qPCR results, among
replicate analyses, increases as the degree of DNA
degradation increases [42]. A260 and fluorescentdye
methods of quantifying intact genomic DNA from IRMM
NK603 reference material DNA provide relatively concor-
dant DNA quantification values [42]. However, the quan-
tification values differ markedly for an identical DNA
extract that has been degraded with its non-degraded
counterpart (Fig. 2). This study revealed that A260 values
overestimate by an average of 20.3% (6.1) and fluorescent
dye methods underestimate by an average of 145.8% (6.0)
the DNA concentration of PCR-amplifiable, intact DNA
extracts of IRMM NK603 reference material [42]. Further-
more, when fluorescentdye methods of DNA quantification
were compared with A260 methods, an average percent
Fig. 2 Time-course study of genomic DNA degradation. DNA from a
single extract was dispensed into separate tubes and heat-treated at 95C
for 060 s. Measurements of DNA concentration by a fluorescentdye
method (PicoGreen) and absorbance at 260 nm were captured at each
time interval on triplicate samples; the mean the standard deviation was
plotted for identical samples. PG PicoGreen. (Reproduced with
permission from [42])
difference of 10.1% (6.3) was reported for intact genomic
DNA, but a much more significant percent difference of
152% (10.3) was reported in degraded genomic DNA.
When Ct (threshold, significant detectable increase in
fluorescence) values were applied to assess the predictability
of qPCR measurements, based upon an A260 method of DNA
quantification, consistently high biases ranging between 28.0
and 42.0% for highly degraded IRMM NK603 genomic
DNA were reported [42]. In comparison, based upon a
fluorescentdye method of DNA quantification, both high
and low biases ranging between 13.0 and 85.0% were
reported [42]. The findings of this work suggest that both
A260 and fluorescentdye methods of DNA quantification
have their limitations in terms of providing accurate and
predictable Ct values in qPCR, especially in DNA samples
that contain significant amounts of degradation.
Summary and future perspectives
A fundamental goal of the agricultural biotechnology
industry, to demonstrate compliance with government-
enforced regulatory mandates, is to generate test results on
identical samples that are comparable, regardless of which
laboratory produces the result or whether it is a DNA-based
or protein-based test. Developing validated methods that
meet appropriate analytical specificities and have calibra-
tion traceability will be the focus of research projects over
the next several years. DNA extraction and quantification
are just two elements in the entire process of generating
analytical measurements by qPCR. At 100% efficiency, the
PCR process is logarithmic-based amplification, with a
doubling of product generated upon completion of each
cycle. Because the process is logarithmic, small fluctuations
in the initial process of PCR result in large fluctuations in
the final analytical measurement. Inhibitors of PCR and
DNA integrity can both affect PCR efficiency, thus
contributing towards measurement uncertainty and the
outcome of the final analytical measurement. To minimize
uncertainty and increase interlaboratory repeatability, dem-
onstrating compliance with predetermined validation
parameters is essential. Once the validation parameters
have been demonstrably verified, and reviewed by impartial
experts, implementing the method into an internationally
recognized standard is essential. This approach will enable
laboratories to demonstrate compliance of regulatory
mandates for both buyers and sellers of these products
and minimize measurement uncertainty.
PCR is generally the technology of choice for laboratories
that detect and quantify levels of GE traits in grains and food
products. To expedite the fair and orderly marketing of these
products, it is prudent to have methods that are sensitive,
rapid, cost-effective, accurate, reliable and reproducible.
1988
Certainly, both implementing standardized methods through
ISO and providing publicly available reference materials/
methods contribute towards harmonization when the presence
of GE traits in grains and oilseeds is being analysed.
Nonetheless, biological factors will always contribute to
measurement uncertainty and must be considered when qPCR
is used to measure the GE content in plant-derived products.
DNA provides a template for the initial steps in qPCR. Studies
show that highly purified, intact DNA, derived from isogenic
plant material, provides the best reproducibility, with predict-
able Ct values when used in qPCR, but the reproducibility
becomes less as DNA becomes degraded or when non-
isogenic samples are used in an analysis [42, 66]. Many
DNA extraction methods are available for the provision of
DNA for identification and quantification of GE traits by
qPCR. DNA of high quality and sufficient yield is obtained
more readily from less processed samples as compared with
highly processed samples. The most commonly used CTAB-
based DNA extraction method has different versions as
described in previous sections of this review and one has to
carefully determine the most appropriate version for each
application. Faster and non-organic-based DNA extraction
methods are desirable. Many commercially available kits are
used for DNA extraction and purification, as shown in
Table 2. One kind of DNA extraction method may not be
suitable for the different kinds of grains/oilseeds, food and
feed products available. The trend is to use combinations of
DNA extraction methods for sufficient cell lysis and isolation
of high-quality DNA that is free from contaminants.
Extraction of high-quality DNA is affected by sample
matrices and biological factors that have been described
previously. For different maize cultivars, variation exists in
the GE copy numbers when equal amounts of DNA are
used. For maize, variation in endosperm and embryo DNA
content as well as homozygosity/heterozygosity will affect
qPCR results. Generally, a greater DNA yield is obtained
from particles of fine size than from particle s of coarse size.
An appropriate DNA extraction procedure must be validated
and demonstrably optimized for yield, quality, integrity and
efficiency in the qPCR. DNA extracted from highly
processed food products generally provides degraded DNA
that might not be suitable for PCR. Amplification of shorter
segments of the target increases the likelihood of successful
qPCR and normalization to an endogenous control becomes
significant for one to have confidence in analytical results.
Some studies question the usefulness of DNA quantification
prior to qPCR, especially in cases where DNA is highly
degraded. In these instances, A260 measurements will not
provide concordant measurements compared with
fluorescent-dye methods and neither method provides highly
predictable Ct values in the qPCR compared with intact
controls. Although agarose gel electrophoresis data are
useful in assessing the extent of DNA degradation, differ-
T. Demeke, G.R. Jenkins
ences in Ct values in the qPCR can be attributable to not
only to the presence of inhibitors but also to differences in
DNA quantification [42].
Isothermal reactions for the amplification of oligonu-
cleotides, an alternative technology to PCR, may overcome
many of the disadvantages associated with PCR [67]. This
technology includes several versions of an exponential
amplification scheme but can operate in a linear amplifica-
tion fashion as well. The reactions depend entirely on the
molecular parameters governing interactions of the mole-
cules in the reaction. The robustness, speed and sensitivity
suggest that it will be useful in the rapid detection of GE
traits and a commercialized version of the technology is
currently being developed.
A final consideration regarding DNA extraction and
quantification is the development of a system which can
provide for greater automation and throughput. The
application of rapid, high-throughput methods coupled
with system automation has the potential to greatly
reduce the time and cost of DNA extraction and
consequently that of overall analysis [14]. Because future
generations of GE products are going to possess more than
one introduced gene, an appropriate approach to deter-
mining whether the material is above or below the
labelling threshold will need to be established. The
variables discussed in this review can hopefully contribute
towards providing standardized guidelines for qPCR
methods, to enable a more systematic and harmonized
approach and facilitate grain marketing.
Acknowledgements Daniel Perry (Canadian Grain Commission,
Winnipeg), Marcia Holden (National Institute of Standards and
Technology), Tandace A. Scholdberg and Donald Kendall (USDA/
GIPSA, Kansas City) are acknowledged for reviewing the paper and
providing useful suggestions.
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