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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, extraction kits, as well as modifications to published
corn and soybean are being grown in the USA, Canada and cetyltrimethylammonium bromide methods. Inhibitors are a
other predominantly grain exporting countries. Although the major obstacle for efficient amplification in qPCR. The types
amount of farmland devoted to production of biotechnology- of PCR inhibitors and techniques to minimize inhibition are
derived crops continues to increase, lingering concerns that discussed. Finally, accurate quantification of DNA for
unintended consequences may occur provide the EU and applications in qPCR is not trivial. Many confounders
most grain-importing countries with justification to regulate contribute to differences in analytical measurements when
these crops. Legislation in the EU requires traceability of a particular DNA quantification method is applied and
grains/oilseeds, food and feed products, and labelling, when different methods do not always provide concordant results
a threshold level of 0.9% w/w of genetically engineered trait on the same DNA sample. How these differences impact
is demonstrated to be present in an analytical sample. The measurement uncertainty in qPCR is considered.
GE content is routinely determined by quantitative PCR
(qPCR) and plant genomic DNA provides the template for Keywords DNA extraction . Sample matrices . DNA
the initial steps in this process. A plethora of DNA extraction quantification . PCR inhibitors . Variability of DNA yield
methods exist for qPCR applications. Implementing stan-
dardized methods for detection of genetically engineered
traits is necessary to facilitate grain marketing. The Interna- Introduction
tional Organization for Standardization draft standard 21571
identifies detergent-based methods and commercially avail- A laudable goal for the future of the agricultural biotech-
able kits that are widely used for DNA extraction, but also nology industry is to identify efficient means of increasing
indicates that adaptations may be necessary depending upon food and feed production, maintaining wholesomeness and
the sample matrix. This review assesses advantages and sustainability, without sacrificing quality or having a
disadvantages of various commercially available DNA detrimental effect on the environment. Many experts in
agricultural biotechnology deem genetically engineered
*Contribution No. 1015 from the Grain Research Laboratory of the (GE) crop plants that confer insect resistance, herbicide
Canadian Grain Commission. tolerance or combinations of these traits as a plausible
T. Demeke (*) approach to meet such objectives. Second-generation
Grain Research Laboratory, Canadian Grain Commission, biotechnology crops will have genes for enhanced nutrition
1404-303 Main Street,
Winnipeg, MB R3C 3G8, Canada
and resistance to abiotic stresses, which will benefit
e-mail: tigst.demeke@grainscanada.gc.ca producers as well as consumers. Biotechnology-derived
crops have been grown in North America since the mid
G. R. Jenkins 1990s. In 2008, biotechnology-derived crops were grown in
United States Department of Agriculture, Grain Inspection,
Packers and Stockyards Administration,
25 countries on 125 million hectares of land [1]. The global
10383 N. Ambassador Dr., acreage of soybean, maize, cotton and canola continues to
Kansas City, MO 64153, USA increase, suggesting affirmation by government officials,
1978 T. Demeke, G.R. Jenkins

research scientists, farmers and the agricultural industry of annexes for extracting and quantifying genomic DNA with
the use and applications of this technology. However, publicly available methods, optimization conditions for
acceptance of GE crop plants into commercial markets has individual laboratory applications is commonly practised
not been without controversy. Although GE crops have [10]. Thus, a plethora of DNA extraction methods exist.
been widely adopted and used on a large scale in the USA, Once DNA has been extracted, prior to PCR, it must be
Canada, Argentina and other grain-exporting countries, quantified. Development of accurate methods to quantify
acceptance of these crops has been more limited in Europe, DNA isolated from plant matrices is not trivial and several
New Zealand and predominantly grain importing countries. methods for the quantification of DNA currently exist. All
The EU implements regulatory regimes and legislative DNA quantification techniques have limitations in their use
mandates based on the process of GE crop production, and application. This review considers the influence of
providing consumer choice through traceability and labelling DNA extraction methods, quantification, and the presence
of GE-derived products (see [2] for EU genetically modified of PCR inhibitors that affect the performance and interpre-
organism regulations). Adventitious or a low-level presence tation of real-time PCR analytical results when testing for
of GE grain in non-GE grain or food/feed products has also the presence of biotechnology-derived traits in food and
been of international concern for countries that abide by the feed products. This review does not cover real-time
Cartagena Protocol on Biosafety. In accordance with this quantitative PCR (qPCR) methods. Relevant references
precautionary approach, living modified organisms must be for real-time qPCR methods are available [1113].
handled, transported, used and released in a manner that
minimizes risks to human health and biological diversity,
irrespective of overwhelming scientific data purporting Commonly used DNA extraction methodstheir effects
efficacy of these products (Article 18.2.a) [3]. Conversely, on DNA quality and PCR
Canada and the USA consider GE products to be substan-
tially equivalent when the nutritional content and a multitude Real-time qPCR is a common method in agricultural
of safety aspects of the finished product are shown to be biotechnology for determining the GE content in food
indistinguishable from those of their non-GE counterparts and feed products. Trace amounts of biotechnology-
[4]. These fundamental differences in philosophies have led derived DNA are routinely analysed by qPCR to verify
to asynchronous regulations worldwide and a potential to the presence or absence of a particular trait, especially
disrupt grain trade unless validated analytical methods are during stages of product development, and for demon-
implemented that (1) monitor the supply chain, (2) accurately strating compliance with legislative mandates. Real-time
certify product content and (3) demonstrate compliance with qPCR amplification is influenced by the quality, purity
international labelling or traceability requirements [5]. and quantity of DNA in a sample. The aim of a nucleic
One of the major challenges facing laboratories that test acid extraction method is to isolate DNA of suitable
for the presence of GE traits in food and feed products is integrity, purity and of sufficient quantity for diagnostic
how to standardize testing procedures. To ensure compa- applications by qPCR [14]. Obtaining DNA of high
rable analytical results by different laboratories, analyses quality is paramount for ensuring confidence in all
should be carried out with validated methods using subsequent steps in the process of generating analytical
standard reference materials such that precision, accuracy, measurements. This section reviews DNA extraction
sensitivity, specificity and robustness can be demonstrably methods that are commonly utilized in agricultural biotech-
optimized. The most commonly used method to determine nology for diagnostic applications.
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 Cetyltrimethylammonium bromide method
GE traits by PCR is dependent upon the specificity and
sensitivity of the amplification procedure as well as the The cetyltrimethylammonium bromide (CTAB) method has
quality and quantity of genomic DNA that is dispensed into been widely used for extraction of DNA from leaves, seeds/
the reaction. grains and processed food/feed samples [10]. The procedure
The appropriate sampling method, sample size, type of is time-consuming and uses hazardous chemicals such as
matrix, biological factors and inhibitors affect the quality phenol and chloroform. The basic CTAB extraction buffer
and quantity of DNA extracted from grain/seed, food and consists of 2% CTAB, 1.4 M NaCl, 20 mM EDTA and
feed samples. A universally accepted sampling method for 100 mM tris(hydroxymethyl)aminomethane (Tris)HCl, pH
the detection and quantification of GE traits does not exist, 8.0. The original protocol [15] involved the use of CTAB
but helpful references on sampling methods are available extraction buffer and chloroform/isoamyl alcohol (24:1) to
[79]. While ISO standard 21571 provides guidance 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 might be trapped within sugar-derived condensation prod-
mercaptoethanol to the CTAB buffer and the use of phenol/ ucts [17, 18]. There are several versions or modifications of
chloroform/isoamyl alcohol (25:24:1) initially and then the CTAB protocol, as indicated in Table 1. It is important
cholorofrom/isoamyl alcohol (24:1) for further purification to note that there are differences in the concentrations of the
[14, 16]. Addition of 10% N-phenacylthiazolium bromide CTAB extraction buffer components used for the different
to the CTAB buffer has been reported to release DNA that 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 T. Demeke, G.R. Jenkins

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

Table 3 Summary of different approaches used for DNA extraction

DNA extraction Cell Protein Polysaccharide Silica Organic Magnetic DNA DNA
method lysis precipitation precipitation gel solvent beads precipitation elution

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 samples of various particle sizes (i.e. flour, meal and grits)
were compared, corn flour with the finest particle size
The quality and quantity of DNA extracted from food products provided the highest yield [33]. This observation can be
tend to decrease with the extent to which the food is processed easily explained by the fact that smaller particles offer a
[25, 28]. Exposure to heat results in fragmentation of larger surface area of exposure to extraction reagents
high molecular weight DNA, and physical and chemical compared with larger particles. Relative GE content quanti-
treatments cause random breaks in DNA strands, reducing the fication could be significantly affected if the analysed food
DNA fragment size [18]. The type of sample used for DNA contains fractions with different particle size distributions.
extraction will influence the quality and quantity of DNA. DNA extractions from fine (4463 M) and coarse (102
Intact and high-quality DNA can be obtained from ground 115 M) corn powders did not, however, influence the DNA
samples of grains and oilseeds. Conversely, DNA extracted copy number estimation [34]. The particle sizes of GE and
from highly processed food and feed samples is generally non-GE ground samples need to be similar for appropriate
more sheared and of low molecular weight. For highly comparison with real-time qPCR. Routine grinding to obtain
sheared DNA, there may not be enough templates available fine particle sizes can be cumbersome and time-consuming,
for PCR and this will have an impact on the limit of detection especially for oil-rich seeds such as canola and soybean.
and quantification. Only minute amounts of DNA can be DNA extracted from samples of ground canola and soybeans
extracted from refined oil, modified starch, etc. and this will that passed through a 1.0-mm sieve have been used
affect the reproducibility and quantification of DNA by PCR. successfully in real-time qPCR assays [16, 35].
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 Biological challenges and variability of DNA yield
processed samples. Studies have been conducted to determine among cultivars, plants and processed foods
whether the percentage of GE product obtained from a
processed food reflected the original percentage of GE Analysis of GE content by qPCR in plant sources involves
product in the raw material [29, 30]. These studies demon- measurement of copy numbers of a trait-specific target gene
strate that in some instances processed foods provide DNA and a taxon-specific, endogenous reference gene. Analysis of
material that is not suitable for measuring GE content by the GE content in plant material by qPCR faces several
qPCR since the copy number of the trait or both the trait- and biological challenges, all of which contribute to measurement
taxon-specific genes were below the quantification limit of 20 uncertainty. For example, when the levels of GE content are
copies of PCR-amplifiable DNA. In other processed food quantified by qPCR, the ratio between the target gene and the
products, where copy number was not limiting, high bias and endogenous reference gene is assumed to be the same for any
a statistically significant difference was observed in the given test or reference sample. However, this assumption is
measured GE content of processed foods (containing mostly not valid under certain circumstances [12, 36]. The Institute
degraded DNA) compared with their raw counterparts for Reference Materials and Measurements (IRMM) maize
(consisting of mostly intact DNA) [30]. event Bt-11 contains two copies of the 35S promoter per
DNA extraction efficiency is unequivocally dependent copy of maize endogenous reference gene [12]. Another
upon particle size. As the particle size in a sample decreases, biological confounder that contributes to measurement
yields increase [31, 32]. When DNA yields amongst corn 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 processed food and feed products contain genomic DNA
million and 2,716 million base pairs per haploid genome, from a multitude of plant and animal sources. Under these
suggesting that different plant cultivars contribute different conditions, when a specified quantity of DNA is added into
copy numbers when equivalent mass amounts are dispensed the PCR, only a small proportion of molecules will contain
into the qPCR [37]. Reports also suggest that DNA yields the target of interest with appropriate length for amplifica-
may not be the same amongst different cultivars, with some tion. To improve the reliability of PCR, normalizing to a
yielding up to 20% less DNA when a CTAB extraction taxon-specific reference (endogenous control) gene becomes
method is used [32, 38]. Ten maize cultivars had ratios of critical [42].
endosperm to total DNA ranging from 36.3 to 59.4%, Several studies evaluating DNA extraction methods to
compared with ratios of embryo to total DNA which ranged characterize yield, quality and predictability in PCR can be
from 38.6 to 59.6% [32]. However, variation in the found in the literature. Identifying the most appropriate
endosperm DNA did not significantly affect the quantifica- method for a particular type of food product, one that
tion of GE content in this study. Characterization studies provides DNA of the highest quality and sufficient yield for
suggest that the maize kernel is made up of 83% endosperm, analysis of a particular food product, is a daunting task [28,
11% embryo, 5% pericarp and 1% tip cap [39]. In a single 30, 40, 43]. Most studies have been designed to characterize
kernel analysis, endosperm plus pericarp contained approx- a representative food type with different extraction methods.
imately 10 times more extractable DNA than did embryo For example, out of nine different extraction methods
[39]. Other reports suggest that about half of the total DNA compared for soybean food samples, five methods provided
extracted from maize kernels originates from endosperm and predominantly degraded DNA, whereas the other four
the other half from embryo [32]. These findings raise methods provided relatively intact DNA but with signifi-
concerns about accurate quantification of GE content using cantly lower yields [43].
qPCR considering that the endosperm consists of two The complexity of detecting GE traits in grains
maternal haploid genomes and one paternal haploid genome, continues to grow as new varieties are introduced into
whereas the embryo consists of one each of a maternal and a the marketplace [44]. The future trend for the agricultural
paternal haploid genome. DNA endoreduplication (duplica- biotechnology industry is to develop cultivars with
tion of the genome without mitosis) in maize also poses a stacked GE traits (e.g. maize). Only single kernel (or
challenge for qPCR. Endoreduplication leads to polyploid- plant) analyses can differentiate between multiple events
ization, in which a single cell contains several copies of the stacked into a single cultivar and a mixture of events
genome [12], and may impact the relative contribution of derived from a mixture of cultivars [45, 46]. However,
embryo and endosperm in ground maize samples. Since single kernel analysis using PCR to detect the presence of
quantification by qPCR cannot distinguish endoreduplication stacked events requires testing of a large number of seeds
or zygosity differences between test and reference samples, per lot and is not economically feasible with currently
the genetic make-up of a test sample and its reference must available PCR technologies.
be the same with respect to zygosity, ploidy and endoredu- Overall, use of certified reference materials, validated
plication status, lest an increase in measurement uncertainty DNA extraction and PCR methods will help to overcome
(i.e. maize kernels homozygous for a given event contribute some of the problems discussed above. A guideline on
more copies of the target gene compared with maize kernels identification and quantification of stacked GE events will
containing the same event in heterozygous state). In the be useful in the near future.
context of transgene identification and quantification, endor-
eduplication amplifies the whole genome and does not alter
the ratio of the copy numbers [36]. Types of PCR inhibitors and methods to reduce
Sufficiently high yields of intact genomic DNA can usually their impact
be obtained from grains, oilseeds or minimally processed food
samples. Conversely, it becomes more challenging to obtain Inefficient PCR amplification may result if an insufficient
high yields of intact genomic DNA from food and feed quantity of DNA of appropriate molecular weight is used or
products that have been subjected to mechanical, thermal, may be due to the presence of inhibitors in the DNA
chemical and enzymatic treatments [14, 40]. When structural extract. Inhibitors in the DNA can reduce the efficiency
damage to DNA occurs, the process appears to be entirely and/or reproducibility of PCR and thus may contribute to
random [41]. Various models have been proposed to describe inaccurate qPCR results. The inhibitory mode of action of
the length of a target DNA sequence and its probability of some of the compounds may be linked with precipitation of
being damaged by random scission [41]. It is common to the DNA, denaturation of DNA or the ability of the
design primers/probe of relatively short length to amplify polymerase enzyme to bind to magnesium ions [47].
short segments of the target sequence. Many highly Inhibitors may kinetically modify PCR amplification by
1984 T. Demeke, G.R. Jenkins

chelating Mg2+, a cofactor for all DNA polymerases, with proteins/enzymes and alter various properties of the
including Taq polymerase, or by binding to target DNA or biopolymers [52]. In potato, a chlorogenic acid concentra-
the DNA polymerase [48]. Enzyme inhibitors may originate tion of 0.240.36 g/L inhibited reverse transcriptase
from either the plant tissue or the reagents used for DNA PCR [53]. An EDTA concentration of 0.5 mM or higher
isolation [49]. has been reported to inhibit PCR (Table 4). At the end of
The major types of PCR inhibitors are listed in Table 4. the extraction process, DNA is usually resuspended in 1
Acidic plant polysaccharides (e.g. dextran sulphate, alginic TE (10 mM Tris-HCl and 1 mM EDTA). The EDTA may
acid) have been reported to be inhibitory, whereas neutral be reduced (e.g. 0.1 or 0.5 TE) or eliminated completely.
polysaccharides (e.g. starch, arabinogalactan) were not Only 10 mM Tris-HCl, pH 8.0 (without EDTA) is also
found to be inhibitory [31, 49, 50]. The presence of being routinely used to resuspend DNA that is used for
0.001% or more dextran sulphate resulted in complete real-time PCR. DNA can also be resuspended in distilled
inhibition of PCR [31]. On the other hand, the presence of sterile water; however, in the absence of stabilizing
4% dextran (a neutral polysaccharide) did not have an compounds the DNA cannot be stored for a long period
effect on PCR. DNA solutions can become viscous owing of time [54].
to the presence of polysaccharides and other substances. It Concentrated protein (e.g. 1% casein hydrolysate) has
is difficult to accurately pipette a solution of highly viscous been reported to inhibit PCR [14, 47]. Ionic detergents such
DNA. Centrifugation of soybean DNA solution [resuspended as SDS are reported to be more inhibitory than non-ionic
in Tris-HCl/EDTA (TE)] at low speed (e.g. 7,000 rpm) for detergents such as Tween 20 and Triton X-100 [47]. CTAB
5.0 min resulted in pelleting out of the viscous material [16]. and SDS concentrations of more than 0.005% inhibited
The supernatant can be used for qualitative PCR, even PCR [49]. The two detergents are usually used early during
though further purification with a kit may be required for DNA extraction, and thus residual amounts are removed
real-time PCR (Table 2). through the subsequent washing steps. Ethanol and 2-
Process-introduced PCR inhibitors include phenol, propanol are routinely used for DNA extraction even
EDTA, sodium chloride and lithium chloride. Phenol at a though they could inhibit PCR. 2-Propanol and ethanol
concentration of more than 0.2% inhibits PCR [49, 51]. are reported to inhibit PCR at concentrations of 1% or more
Phenolic compounds from the sample or carried over from [49]. Drying the DNA pellet prior to resuspension removes
DNA purification can inhibit PCR by binding to or residual ethanol and 2-propanol.
denaturing the polymerase [48]. Generally, plant phenolic Powder materials from gloves worn in the laboratory
compounds (e.g. chlorogenic acid, caffeic acid) can react have been shown to inhibit PCR. Washing gloves or using

Table 4 Examples of PCR inhibitors reported in the literature and methods to minimize inhibition

Inhibitors Description and inhibitory concentration for PCR Methods to minimize inhibition

CTAB 0.005% (w/v) [49, 51]; 0.01% [47] 70% ethanol wash
EDTA 0.5 mM [49, 51]; 1 mM [47] 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)
Ethanol >1% (v/v) [49] Dry pellet and resuspend
Fat Lipase or hexane treatment and chloroform extraction [14]
Isopropanol >1% (v/v) [49] Dry pellet and resuspend [14]
Phenol >2% (v/v, [49]; >0.2% [51] Use PVP, PVP/ammonium acetate [14]
Chlorogenic acidplant phenol (0.240.36 g/L)inhibited Incorporation of 1.2% citric acid at the DNA extraction step
PCR in potato [53] neutralized the inhibitory effect of chlorogenic acid
Polysaccharides Acidic polysaccharides such as dextran sulphate Use CTAB buffer and chloroform extraction. Treatment with
are inhibitory [14, 50] enzymes such as pectinase, cellulase, hemicellulase and
Dextran sulphate: >0.1% [51]; 0.001% [30] -amylase can be used to remove polysaccharides High
Pectin: >0.5% [49] salt precipitation
Xylan: >0.0025% [51]
Protein 1% casein hydrolysate in PCR mixture caused inhibition [47]. Use SDS, CTAB or guanidinium buffers, proteinase K
SDS 0.005% (w/v, [49] Wash with 70% ethanol
Sodium acetate 5 mM [49] Wash with 70% ethanol
Sodium chloride 25 mM [49] 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 quantification measurement by UV spectrophotometry. As
[48]. Commercially available kits are being widely used for dsDNA becomes more highly denatured, the A260 value
purification of DNA extracted by CTAB and other methods routinely increases accordingly. The observed A260 increase
(Table 2). Some of the kits are used for both extraction and in degraded DNA samples is reportedly due to hyperchromic
purification of DNA. Dilution of the DNA helps to reduce effects wherein strand separation and base pair unstacking
the inhibitor concentration and enhance PCR efficiency; contribute to an increase in the absorbance signal above that
however, a lower DNA concentration may decrease PCR of purely double stranded DNA [58, 59]. When DNA is
sensitivity. An inhibition test using either internal controls denatured, interactions between base pairs can become
or evaluation of the linearity of the calibration curves disrupted as the double-helix structure of DNA dissociates
should be performed to determine the suitability of the into single strands. As a result, the bases assume a larger
extracted DNA for real-time PCR amplification [12, 19]. 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 quantification technologies DNA concentrations precisely and accurately. Inhomogeneity
may occur when DNA is obtained in a highly concentrated
Prior to qPCR, stock DNA extracts are commonly form, predominantly of high molecular weight [57]. Quanti-
quantified and diluted so that all reference and test samples fication by UV spectrophotometry generally requires rela-
contain identical amounts of DNA. Accurate determination tively large amounts of DNA, typically 5.050 g DNA/mL
of DNA concentration in a sample is a critical component per sample A260 0:1  1:0 OD units, compared with
for analysis of GE traits by qPCR. It may be of little value fluorescent-dye methods, which typically require 0.03
to have a method for measuring DNA concentrations with a 0.25 g DNA/mL [57, 60]. In summary, accuracy and
high level of accuracy if the extracts themselves do not precision of DNA quantification by UV spectrophotometry
yield pure DNA. In general, DNA quantification prior to can be influenced by (1) the size distribution of DNA in
qPCR increases confidence in negative PCR results in solution, (2) the ratio of ssDNA and dsDNA and (3) the
diagnostics where insufficient target DNA could otherwise presence of other components that absorb at 260 nm, such as
be interpreted as a false-negative result. The most com- RNA and single nucleotides.
monly used techniques for DNA quantification include UV
spectrophotometry, fluorometry, chemiluminesence and
agarose gel electrophoresis. These are discussed in more Fluorometry
detail in the following sections.
Fluorometry is a useful, rapid, relatively inexpensive method
for quantifying dsDNA prior to qPCR. For measurement of
UV spectrophotometry the concentration of DNA, fluorometry has a sensitivity
similar to that of chemiluminescence (as described later) and
Concentrations of nucleic acid solutions, for PCR and other both techniques are more sensitive than spectrophotometry.
molecular biology applications, are commonly quantified Preparations of serially diluted standards and test samples are
by measuring the absorbance at 260 nm (A260). For DNA incubated with a fluorescent dye such as Hoechst 33258 or
that is predominantly intact, a solution with an A260 value PicoGreen reagent. Fluorescence values of the standards are
that equals one optical density (OD) unit corresponds to a used to construct a linear regression against which the
sample that contains 50 g DNA/mL. For DNA that is concentrations of the test samples are estimated by interpola-
largely degraded or consists of single-stranded DNA tion. The linear concentration range for DNA quantification
(ssDNA), an A260 value of 1 OD unit was found to extends over 4 orders or magnitude (25 pg/mL to 1 g/mL)
correspond to 38 g DNA/mL [55]. For proteins in using a single dye concentration. Assay linearity is maintained
solution, an A280 value that equals 1 OD unit corresponds in the presence of proteins, urea, ethanol and chloroform but
to a protein level of 1 g/mL [56]. Protein contamination of not ionic detergents.
DNA is routinely assessed by comparing the A260/A280 Factors that influence accuracy and precision of DNA
ratio; with a value of 1.7 or greater being acceptable [57]. quantification using fluorometry include (1) the size distribu-
Contaminants including RNA, single-stranded nucleic tion of DNA in solution, (2) exclusivity of dyes (i.e. some
acids and phenols contribute to the total signal in an A260 dyes are more specific for dsDNA), (3) sensitivity of different
measurement and are not discernible from double-stranded dyes and their optimal working concentration ranges, (4)
DNA (dsDNA). The degree to which DNA degradation DNA integrity, (5) sensitivity of the assay to photobleaching
occurs and the ratio of ssDNA to dsDNA may vary from and (6) DNA purity [61]. Hoechst 33258 requires high salt
extract to extract, suggesting uncertainty in any DNA concentrations to detect dsDNA in the presence of RNA and
1986 T. Demeke, G.R. Jenkins

low salt concentrations to detect dsDNA in the presence of tion by chemiluminescence is less affected by contaminants
ssDNA, suggesting that two different assay solutions are than spectrophotometry and fluorometry.
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. Agarose gel electrophoresis
PicoGreen tends to show greater dsDNA:RNA selectivity
compared with Hoechst 33258 [61]. The PicoGreen assay Agarose gel electrophoresis is a commonly used method to
is suitable for quantifying dsDNA prior to PCR and for separate DNA and RNA molecules of different sizes but can
quantifying post-PCR products but is not suitable for thermal also be used to assess DNA quality and provides useful
cycling as when melting steps are included. PicoGreen dye information regarding the amount of DNA in a solution. Since
is sensitive to salts, especially divalent cations and ionic DNA has a consistent charge-to-mass ratio, the major factor
detergents. Some reports suggest as there is as much as that influences its migration through a gel matrix is size [57].
94.6% decrease in signal intensity with PicoGreen when Smaller DNA molecules migrate through gels at a faster rate
genomic DNA contains 0.005% CTAB detergent [60]. 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-
Chemiluminescence trations of agarose facilitate separation of smaller DNA
molecules, whereas lower agarose concentrations allow
Chemiluminescence is a highly sensitive method, designed resolution of larger DNA molecules. Agarose concentrations
for the specific detection of linear, dsDNA [62]. When this of 0.81% are typically used for quantification of genomic
method of DNA quantification is used, chromosomal plant DNA. Once electrophoresis is complete, the DNA in the gel
DNA must be reduced to fewer than 6,000 base pairs in is visualized by staining with a fluorescent dye (e.g. ethidium
length by an appropriate restriction enzyme digestion prior bromide, GelRed, SYBR green), followed by illumination
to the assay being performed [63]. When high molecular with UV light. Ethidium bromide intercalates between bases
weight genomic DNA is used in this assay, the light output of nucleic acids and allows very convenient detection of
does not produce the same signal as an equivalent mass of DNA fragments in gels. The fluorescence of the dyeDNA
smaller fragmented DNA. Restriction enzymes producing complex is greater than that of the unbound dye. The
blunt-ended, 5 overhang or 3 overhang DNA provide presence of an intense, high molecular weight band in the gel
results concordant with A260 measurements [63]. The indicates intact genomic DNA with minimal degradation.
measurement is based upon two coupled enzymatic Degraded or sheared DNA and RNA contamination can be
reactions that produce a light signal proportional to the visualized as a smear towards the lower molecular weight
amount of linear dsDNA in the sample (see Fig. 1) [63]. portion of the gel [42]. DNA concentrations can be estimated
DNA is depolymerized, the deoxynucleotides are converted semiquantitatively by running serially diluted DNA, of
to ATP and luciferase enzyme is added, which allows for known concentration, and visually comparing the intensity
ATP quantification. The amount of ATP is measured by a of the fragment bands with the intensity of the bands of test
luciferase/luciferin reagent. The light signal produced in the samples of unknown concentrations.
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 Limitations of DNA quantification technologies
output measured from standards. The linear range of this and effects of degraded compared with intact DNA
assay is between 20 and 1,000 pg of total DNA per on qPCR
reaction. The amount of ATP generated peaks after
approximately 10 min. The enzyme ATPase can signifi- The purpose of an extraction method is to obtain highly
cantly affect the accuracy of this assay system. Quantifica- pure DNA that provides an acceptable quantity of material

Fig. 1 Quantification of DNA using a chemiluminescence system. dsDNA double-stranded DNA. (Reproduced with permission from Promega)
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 difference of 10.1% (6.3) was reported for intact genomic
has been extracted, the level of DNA degradation that DNA, but a much more significant percent difference of
occurs varies significantly and is contingent upon the 152% (10.3) was reported in degraded genomic DNA.
sample source, processing, extraction protocol and other When Ct (threshold, significant detectable increase in
contributing factors. Also, the degree of degradation fluorescence) values were applied to assess the predictability
between reference and test sample DNA can vary signifi- of qPCR measurements, based upon an A260 method of DNA
cantly. Reports suggest that when DNA is highly degraded, quantification, consistently high biases ranging between 28.0
and the targets for the GE and reference genes differ and 42.0% for highly degraded IRMM NK603 genomic
significantly in size, the reference assay may not normalize DNA were reported [42]. In comparison, based upon a
the GE content correctly [64]. Agarose gel electrophoresis fluorescentdye method of DNA quantification, both high
enables visualization, to some extent, of the degree of DNA and low biases ranging between 13.0 and 85.0% were
degradation within a specified sample. Generally, levels of reported [42]. The findings of this work suggest that both
DNA degradation are more pronounced in processed foods A260 and fluorescentdye methods of DNA quantification
and feeds as compared with IRMM reference material [64, have their limitations in terms of providing accurate and
65]. In theory, the final qPCR measurement assigned to a predictable Ct values in qPCR, especially in DNA samples
particular sample should be independent of the degree of that contain significant amounts of degradation.
DNA degradation since GE target sequence and taxon-
specific sequences degrade congruently. In practice, some
reports suggest that variability in qPCR results, among Summary and future perspectives
replicate analyses, increases as the degree of DNA
degradation increases [42]. A260 and fluorescentdye A fundamental goal of the agricultural biotechnology
methods of quantifying intact genomic DNA from IRMM industry, to demonstrate compliance with government-
NK603 reference material DNA provide relatively concor- enforced regulatory mandates, is to generate test results on
dant DNA quantification values [42]. However, the quan- identical samples that are comparable, regardless of which
tification values differ markedly for an identical DNA laboratory produces the result or whether it is a DNA-based
extract that has been degraded with its non-degraded or protein-based test. Developing validated methods that
counterpart (Fig. 2). This study revealed that A260 values meet appropriate analytical specificities and have calibra-
overestimate by an average of 20.3% (6.1) and fluorescent tion traceability will be the focus of research projects over
dye methods underestimate by an average of 145.8% (6.0) the next several years. DNA extraction and quantification
the DNA concentration of PCR-amplifiable, intact DNA are just two elements in the entire process of generating
extracts of IRMM NK603 reference material [42]. Further- analytical measurements by qPCR. At 100% efficiency, the
more, when fluorescentdye methods of DNA quantification PCR process is logarithmic-based amplification, with a
were compared with A260 methods, an average percent 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
Fig. 2 Time-course study of genomic DNA degradation. DNA from a and minimize measurement uncertainty.
single extract was dispensed into separate tubes and heat-treated at 95C PCR is generally the technology of choice for laboratories
for 060 s. Measurements of DNA concentration by a fluorescentdye that detect and quantify levels of GE traits in grains and food
method (PicoGreen) and absorbance at 260 nm were captured at each
time interval on triplicate samples; the mean the standard deviation was
products. To expedite the fair and orderly marketing of these
plotted for identical samples. PG PicoGreen. (Reproduced with products, it is prudent to have methods that are sensitive,
permission from [42]) rapid, cost-effective, accurate, reliable and reproducible.
1988 T. Demeke, G.R. Jenkins

Certainly, both implementing standardized methods through ences in Ct values in the qPCR can be attributable to not
ISO and providing publicly available reference materials/ only to the presence of inhibitors but also to differences in
methods contribute towards harmonization when the presence DNA quantification [42].
of GE traits in grains and oilseeds is being analysed. Isothermal reactions for the amplification of oligonu-
Nonetheless, biological factors will always contribute to cleotides, an alternative technology to PCR, may overcome
measurement uncertainty and must be considered when qPCR many of the disadvantages associated with PCR [67]. This
is used to measure the GE content in plant-derived products. technology includes several versions of an exponential
DNA provides a template for the initial steps in qPCR. Studies amplification scheme but can operate in a linear amplifica-
show that highly purified, intact DNA, derived from isogenic tion fashion as well. The reactions depend entirely on the
plant material, provides the best reproducibility, with predict- molecular parameters governing interactions of the mole-
able Ct values when used in qPCR, but the reproducibility cules in the reaction. The robustness, speed and sensitivity
becomes less as DNA becomes degraded or when non- suggest that it will be useful in the rapid detection of GE
isogenic samples are used in an analysis [42, 66]. Many traits and a commercialized version of the technology is
DNA extraction methods are available for the provision of currently being developed.
DNA for identification and quantification of GE traits by A final consideration regarding DNA extraction and
qPCR. DNA of high quality and sufficient yield is obtained quantification is the development of a system which can
more readily from less processed samples as compared with provide for greater automation and throughput. The
highly processed samples. The most commonly used CTAB- application of rapid, high-throughput methods coupled
based DNA extraction method has different versions as with system automation has the potential to greatly
described in previous sections of this review and one has to reduce the time and cost of DNA extraction and
carefully determine the most appropriate version for each consequently that of overall analysis [14]. Because future
application. Faster and non-organic-based DNA extraction generations of GE products are going to possess more than
methods are desirable. Many commercially available kits are one introduced gene, an appropriate approach to deter-
used for DNA extraction and purification, as shown in mining whether the material is above or below the
Table 2. One kind of DNA extraction method may not be labelling threshold will need to be established. The
suitable for the different kinds of grains/oilseeds, food and variables discussed in this review can hopefully contribute
feed products available. The trend is to use combinations of towards providing standardized guidelines for qPCR
DNA extraction methods for sufficient cell lysis and isolation methods, to enable a more systematic and harmonized
of high-quality DNA that is free from contaminants. approach and facilitate grain marketing.
Extraction of high-quality DNA is affected by sample
matrices and biological factors that have been described
Acknowledgements Daniel Perry (Canadian Grain Commission,
previously. For different maize cultivars, variation exists in
Winnipeg), Marcia Holden (National Institute of Standards and
the GE copy numbers when equal amounts of DNA are Technology), Tandace A. Scholdberg and Donald Kendall (USDA/
used. For maize, variation in endosperm and embryo DNA GIPSA, Kansas City) are acknowledged for reviewing the paper and
content as well as homozygosity/heterozygosity will affect providing useful suggestions.
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 References
and demonstrably optimized for yield, quality, integrity and
efficiency in the qPCR. DNA extracted from highly 1. ISAAA (2009) ISAAA brief 39-2008: executive summary. http://
processed food products generally provides degraded DNA www.isaaa.org/resources/publications/briefs/39/executivesummary/
that might not be suitable for PCR. Amplification of shorter default.html
2. Davison J, Bertheau Y (2008) Cereal Foods World 53:186196
segments of the target increases the likelihood of successful 3. Convention on Biological Diversity (2000) Protocol on
qPCR and normalization to an endogenous control becomes biosafety. Text of the protocol. http://www.cbd.int/biosafety/
significant for one to have confidence in analytical results. protocol.shtml
Some studies question the usefulness of DNA quantification 4. Hobbs JE, Gaisford JD, Isaac GE, Kerr WA, Klein KK (2002) In:
Agricultural biotechnologies: new avenues for production, con-
prior to qPCR, especially in cases where DNA is highly sumption and technology transfer. Sixth international ICABR
degraded. In these instances, A260 measurements will not conference, Ravello, Italy
provide concordant measurements compared with 5. Giroux GR (2008) Sample preparation and extraction. http://
fluorescent-dye methods and neither method provides highly gmoglobalconference.jrc.ec.europa.eu/2008/Presentations/Giroux
%20-%20Intl%20testing%20mtg_2008_final.pdf
predictable Ct values in the qPCR compared with intact 6. Grothaus DG, Bandla B, Currier T, Giroux GR, Jenkins GR,
controls. Although agarose gel electrophoresis data are Lipp M, Shan G, Stave JW, Pantella V (2006) J AOAC Int
useful in assessing the extent of DNA degradation, differ- 89:913928
Influence of DNA extraction methods, PCR inhibitors and quantification methods on real-time PCR assay of biotechnology-derived traits 1989

7. Fagan J (2004) In: Ahmed FE (ed) Testing of genetically 36. Holst-Jensen A, De Loose M, van den Eede G (2006) J Agric
modified organisms in food. Food Products, New York, pp Food Chem 54:27992809
163220 37. Arumuganathan K, Earle ED (1991) Plant Mol Biol Rep 9:202
8. Freese LD (2004)) In: Ahmed FE (ed) Testing of genetically 218
modified organisms in food. Food Products, New York, pp 55 38. Heinemann JA, Sparrow AD, Traavik T (2004) Trends Biotechnol
75 22:331336
9. Laffont JL, Remund KM, Wright D, Simpson RD, Grgoire S 39. Papazova N, Malef A, Degrieck I, van Bockstaele, De Loose M
(2005) Seed Sci Res 15:197204 (2005) Seed Sci Technol 33:533542
10. International Organization for Standardization (2005) ISO 40. Mafra I, Silva SA, Moreira EJMO, Ferreira de Silva CS, Beatriz M,
21571:2005. Foodstuffsmethods of analysis for the detection Oliveira PP (2008) Food Control 19:11831190
of genetically modified organisms and derived productsnucleic 41. Deagle BE, Eveson JP, Jarman SN (2006) Front Zool 3:11
acid extraction. http://www.iso.org/iso/iso_catalogue/catalogue_ 42. Shokere LA, Holden MJ, Jenkins GR (2009) Food Control
tc/catalogue_detail.htm?csnumber=34616 20:391401
11. Ahmed FE (ed) (2004) Testing of genetically modified organisms 43. Zimmerman A, Luthy J, Pauli U (1998) Z Lebensm Unters Forsch A
in food. Food Products, New York 207:8190
12. Lipp M, Shillito R, Giroux R, Spiegelhalter F, Charlton S, Pinero 44. Layton DT, Spiegelhalter F (2008) How grain companies are
D, Song P (2005) J AOAC Int 88:136155 managing the challenges posed by stacked events in meeting the
13. Miraglia M, Berdal KG, Brera C, Corbisier P, Holst-Jensen A, global regulatory and commercial requirements for non-GM corn
Kok EJ, Marvin HJP, Schimmel H, Rentsch J, van Rie JPPF, shipmentsA comparison of methods in current use. http://
Zagon J (2004) Food Chem Toxicol 42:11571180 gmoglobalconference.jrc.ec.europa.eu/menu.htm
14. Terry CF, Harris N, Parkes HC (2002) J AOAC Int 85:768774 45. Akiyama H, Watanabe T, Wakabayashi K, Nakade S, Yasui S,
15. Doyle JJ, Dole JL (1987) Phytochem Bull 19:1115 Sakata K, Chiba R, Spiegelhalter F, Hino A, Maitani T (2005)
16. Demeke T, Ratnayaka I, Phan A (2009) J AOAC Int 92:11361144 Anal Chem 77:74217428
17. Bernardo GD, Galderisi U, Cipollaro M, Cascino A (2005) 46. Akiyama H, Sakata K, Kondo K, Tanaka A, Liu MS, Oguchi T,
Biotechnol Prog 21:546549 Furui S, Kitta K, Hino A, Teshima R (2008) J Agric Food Chem
18. Bernardo GD, Gaudio SD, Galderisi U, Cascino A, Cipollaro M 56:19771983
(2007) Biotechnol Prog 23:297301 47. Rossen L, Nrskov P, Holmstrm K, Rasmussen OF (1992) Int J
19. Corbisier P, Broothaerts W, Gioria S, Schimmel H, Burns M, Food Microbiol 17:3745
Baoutina A, Emslie KR, Furi S, Kurosawa Y, Holden MJ, Kim H-H, 48. Wilson IG (1997) Appl Environ Microbiol 63:37413751
Lee Y-M, Kawaharasaki M, Sin D, Wang J (2007) J Agric Food 49. Peist R, Honsel D, Twieling G, Lffert D (2001) Qiagen News 3:79
Chem 55:32493257 50. Demeke T, Adams RP (1992) Biotechniques 12:332334
20. CRL-GMFF Community Reference Laboratory for GM Food and 51. Popping B (2008) Aspects of modular sampling preparation
Feed (2009) Pioneer. http://gmo-crl.jrc.ec.europa.eu/summaries/ protocols for various matrices. http://gmoglobalconference.jrc.ec.
356043-5_DNAExtr_report.pdf europa.eu/DetailedProgramme.htm
21. CRL-GMFF Community Reference Laboratory for GM Food and 52. Rohn S, Rawel HM, Kroll J (2002) J Agric Food Chem
Feed (2007) Syngenta. http://gmo-crl.jrc.ec.europa.eu/summaries/ 50:35663571
MIR604_DNAExtr.pdf 53. Singh RP, Singh M, King RR (1998) J Virol Methods 74:231
22. Dellaporta SL, Wood J, Hicks JB (1983) Plant Mol Biol Rep 235
1:1921 54. Duncan E, Setzke E, Lehmann J (2003) General considerations for
23. CRL-GMFF Community Reference Laboratory for GM Food purification of genomic DNA. http://www1.qiagen.com/literature/
and Feed (2007) Bayer Crop Science. http://gmo-crl.jrc.ec.europa. qiagennews/weeklyarticle/Dec03/e48/default.aspx?
eu/summaries/A2704-12_soybean_DNAExtr_report.pdf 55. Cavaluzzi MJ, Borer PN (2004) Nucleic Acids Res 32:19
24. Kim CS, Lee CH, Shin JS, Chung YS, Hyung NI (1997) Nucleic 56. Stenesh J (1984) Experimental biochemistry. Allyn and Bacon,
Acids Res 25:10851086 Needham Heights
25. Smith DS, Maxwell PW, De Boer SH (2005) J Agric Food Chem 57. Sambrook J, Russell DW (2001) Molecular cloning, a laboratory
53:98489859 manual, 3rd edn. Cold Spring Harbor Laboratory Press, Cold
26. Smith DS, Maxwell PW (2007) Food Control 18:236242 Spring Harbor
27. Cankar K, Stebih D, Dreo T, Zel J, Gruden K (2006) BMC 58. Ageno M, Dore E, Frontali C (1969) Biophys J 9:12811311
Biotechnol 6:37 59. Freifelder D (1978) The DNA molecule, structure and properties.
28. Peano C, Samson MC, Palmieri L, Gulli M, Marmiroli N (2004) J Freeman, San Francisco
Agric Food Chem 52:69626968 60. Holden M, Haynes R, Rabb S, Satija N, Yang K, Blasic JR (2009)
29. Vatilingom M, Pijnenburg H, Gendre F, Brignon P (1999) J Agric J Agric Food Chem 57:72217226
Food Chem 47:52615266 61. Singer VL, Jones LJ, Yue ST, Haugland RP (1997) Anal Biochem
30. Yoshimura T, Kuribara H, Kodama T, Yamata S, Futo S, 249:228238
Watanabe S, Aoki N, Lizuka T, Akiyama H, Maitani T, Naito S, 62. Walsh PS, Varlaro J, Reynolds R (1992) Nucleic Acids Res
Hino A (2005) J Agric Food Chem 53:20602069 20:50615065
31. Holden MJ, Blasic JR, Bussjaeger L, Kao C, Shokere LA, Kendall 63. Kinney J, Leippe D, Lewis K, Lyke B, Mandrekar M, Schultz J
DC, Freese L, Jenkins GR (2003) J Agric Food Chem 51:2468 (1999) The DNAQuant DNA quantitation system for sensitive
2474 detection of dsDNA. http://www.promega.com/pnotes/73/
32. Trifa Y, Zhang D (2004) J Agric Food Chem 52:10441048 8235_03/8235_03.html
33. Moreano F, Busch U, Engel K-H (2005) J Agric Food Chem 64. Chen YW, Ge Y, Xu B (2005) J Agric Food Chem 53:10239
53:99719979 10243
34. Charles D, Broeders S, Corbisier P, Trapmann S, Schimmel H, 65. Hupfer CH, Hortzel H, Sachse K, Engel KH (1999) Z Lebensm
Linsinger T, Emons H (2007) J Agric Food Chem 55:3258 Unters Forsch A 206:203207
3267 66. Scholdberg TA, Norden TD, Nelson DD, Jenkins GR (2009) J
35. Demeke T, Ratnayaka I (2008) Food Control 19:893897 Agric Food Chem 57:29032911
1990 T. Demeke, G.R. Jenkins

67. Van Ness J, Van Ness LK, Galas DJ (2003) Proc Natl Acad Sci 73. Rizzi A, Panebianco L, Giaccu D, Sorlini C, Daffonchio D (2003)
USA 100(8):45044509 Ital J Food Sci 15:499510
68. Murray MG, Thompson WF (1980) Nucleic Acids Res 8:43214325 74. Minegishi Y, Kurosawa Y, Nishikawa C, Doi N, Kanayama S,
69. Lipp M, Brodmann P, Pietsch K, Pauwels J, Anklam E (1999) J Kodama T, Kasahara M, Matsuoka T, Watanabe T, Akiyama H,
AOAC Int 82:923928 Teshima R, Mano J, Furui S, Hino A, Kitta K (2008) Evaluation
70. Niu C, Kebede H, Auld DL, Woodward JE, Burow G, Wright RJ of a new DNA extraction method for PCR detection of genetically
(2008) Afr J Biotechnol 7:28182822 modified soybean. http://gmoglobalconference.jrc.ec.europa.eu/
71. CRL-GMFF Community Reference Laboratory for GM Food and menu.htm
Feed (2007) Monsanto. http://gmo-crl.jrc.ec.europa.eu/summaries/ 75. Jasbeer K, Ghazali FM, Cheah YK, Son R (2008) Comparison of
RT73_DNAExtr_report.pdf seven methods for the extraction of DNA from compound feed
72. Chhalliyil P, Fagan J, Schoel B (2008) Superior performance of fast ID samples for the purpose of GMO analysis. http://gmoglobalcon
DNA extraction kit in isolation of high quality DNA from food and ference.jrc.ec.europa.eu/menu.htm
feed samples for real-time qPCR analyses exemplified with soy and 76. Tengel C, Schler P, Setzke E, Balles J, Sprenger-Hauels M
rice products. http://gmoglobalconference.jrc.ec.europa.eu/menu.htm (2001) Biotechniques 31:426429