Botanical Studies (2006) 47: 1-11.
*
Corresponding author: E-mail: mbmtchan@gate.sinica.edu.tw; Tel: 886-2-26516194; Fax: 886-2-26511164.
Validation of cDNA microarray as a prototype for
throughput detection of GMOs
Tzy-Li CHEN
1
, SANJAYA
2
, Venkatesh PRASAD
2
, Ching-Hua LEE
2,3
, Kuang-Hung LIN
4
,
Lih-Ching CHIUEH
5
, and Ming-Tsair CHAN
2,
*
1
Department of Food Science and Technology, Tung-Fang Institute of Technology, KaoHsiung 829, Taiwan
2
Institute of BioAgricultural Sciences, Academia Sinica, Taipei 115, Taiwan
3
Graduate Institute of Biotechnology, Chinese Culture University, Taipei 111, Taiwan
4
Department of Horticulture, Chinese Culture University, Taipei 111, Taiwan
5
Bureau of Food and Drug Analysis, Department of Health Executive Yuan, Taipei 115, Taiwan
(Received November 16, 2004; Accepted August 9, 2005)
ABSTRACT
. As a step towards developing an efficient genetically modified organism (GMO) detection
system, the present investigation proposes microarray as a prototype for high-throughput detection of pure
GMO samples. Common T-DNA regions of the expression cassette such as 35S promoter, 35S terminator,
nopaline synthase terminator (NOSt), hygromycin, and kanamycin selection marker genes were detected in
the transgenic rice, tomato, and potato developed in our laboratory. For proof-of-concept purposes, market-
introduced GM potato, GM maize, and cornflakes were screened. The non-GM potato and maize control
detected only the presence of endogenous genes while the GM targets detected the presence of transgenic
genes such as CaMV 35S promoter, 35S terminator, NOSt and nptII, pat, cp4 epsps, cry1 Ab genes on chips.
Moreover, it was observed that the sensitivity of this system for serially diluted GM potato tubers was up
to 0.01-0.002% of the mass fraction. Due to its high accuracy and speed, it is believed that the microarray
detection system will play an important role in routine, high-throughput detection of pure GMO samples in
the future.
Keywords: cDNA microarray; Genetically modified organisms (GMO); GM potato; GM maize.
Abbreviations: 35Sp, promoter from the cauliflower mosaic virus; 35St, CaMV35S poly (A) signal; aadA,
streptomycin-resistance; Actin, rice actin gene; BAR, gene coding for a phosphinothricin acetyltransferase
from Streptomyces hygroscopicus; Bt11, specific gene of Bt11 (Novartis); b-tubulin: tomato b-tubulin gene;
CBH351, specific gene for CBH351 (StarLink, AgrEvo); CryIAb, delta-endotoxin from Bacillus thuringi-
ensis subsp. Kurstaki; EPSPS, 5-enolpyruvylshikimate-3-phosphate synthase from Agrobacterium tume-
faciens strain CP4; GA21, specific gene of GA21 (Monsanto); GFP, green fluorescent protein gene; GUS,
b-glucuronidase gene; HPT, hygromycin phosphotransferase gene; ivr, maize invertase gene; LUC, luciferase
gene; LE, soybean legumin protein gene; NOSt, terminator of nopaline synthase gene from Agrobacterium
tumefaciens; NPT II, neomycin phosphotransferase gene; Ocst, octopine synthase terminator; tmlt, transcrip-
tion terminator of a tumor morphology large gene from Agrobacterium tumefaciens; T25, specific gene of
T25 Libery (AgrEvo); E35S, enhanced CaMV35S promoter; B P, bacterial promoter; bla, beta lactamase; pat,
phosphinothricin-N-acetyltransferase from S. viridochromogenes; bla, beta-lactamase gene; conveys resistance
to beta-lactam antibiotics; from Tn3; Zmhsp70, maize HSP 70 intron; Cpsp, chloroplast transit peptide from A.
thaliana EPSPS gene; P-ract1/ract1, P-ract1/ract1 intron; Actin 1, rice actin I promoter; RuBisco-sp, ribu-
lose-1,5-bisphosphate carboxylase oxygenase derived chloroplast transit peptide sequence; AI, Actin 1 intron
sequence; IVS 2, IVS2 intron from the maize alcohol dehydrogenase gene; IVS6, IVS6 intron from the maize
alcohol dehydrogenase gene.
MOLECULAR BIOLOGY
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Botanical Studies, Vol. 47, 2006
INTRODUCTION
Recombinant DNA technology has hastened crop
improvement strategies through precise genetic
manipulation involving plant specific processes.
Previously, such improvements were only possible
through cumbersome crosses between crops and their
wild-relatives. However, the introduction of genetically
modified food, certified by legally competent authorities
as food or feed since 1994 (Bertheau et al., 2002) has
caused unforeseen apprehension among the public, due
to a lack of stringent biosafety regulations and reported
allergic responses to genetically modified food by humans
(CDC, 2001; Soboleva et al., 2003). As a step towards
addressing controversies pertaining to GM foods, GMO
regulation bodies in different parts of Europe and Brazil
are considering whether to demarcate genetic engineering
technology from other technological processes (Oda and
Soares, 2000). A bill passed in the California senate, AB
2962, requires the labeling of genetically engineered fish
in supermarkets (Campaign, 2002). China is undertaking
extensive field trials of transgenic crops to implement new
rules on GMO, according to the China Daily, 21 March
2002.
GMO technology for developing and introducing
genetically modified food, from a social standpoint, has
necessitated GMO detection primarily to address concerns
related to safety, but also those related to ethics, sociology,
and intellectual property rights. It is further intended to
keep the consumer’s choice on a par with non-GM foods
and facilitate the monitoring of the measurable effects of
gene contamination in indigenous species, by competent
authorities. As a bio-safety measure it is also important
for testing the homogeneity of imported seeds, grain,
commodities and organic products of conventionally
grown crops imported from other countries. Detection
methods primarily need to look for genes engineered
in crops, such as the promoter sequence, foreign genes
coding for a desired trait, and the terminator sequence
which functions as a stop signal in the reading frame.
Reported GMO detection methods include
immunoassay to detect proteins and PCR analysis to
detect DNA. Immunoassays lack the advantage of
screening a large number of samples in a shorter time
(Brett et al., 1999). The official Swiss method (PCR
based) uses detection of cauliflower mosaic virus promoter
(35S promoter) and the nopaline synthase terminator (nos
3’), which are present in most GM crops approved today
(Hardegger et al., 1999). However, this screening method
has limited sensitivity and specificity (Brodmann et al.,
2002). Most of the PCR experiments enable detection
of one gene/promoter (e.g., CaMV 35S) in each sample
at a time. However, with more and more GM foods and
products reaching the market, a cost effective and high-
throughput method of detecting GM products is urgently
needed. The microarray method is time-result oriented,
enabling detection of more than one gene, including
promoter, foreign gene, endogenous control gene, reporter
gene, selection marker gene, and stop sequence.
DNA microarrays are analytical systems allowing the
simultaneous detection of many nucleic acid sequences (up
to thousands) in a sample. This technology is based on the
hybridization of complementary probe and target genes /
cDNAs. The cDNAs or PCR products are arranged on a
solid support (e.g. glass slide) as probes. The probes on
the array are then hybridized with the fluorescently labeled
PCR products or genes form GM/non-GM samples (called
targets). Laser scanning analysis then reveals the presence
of labeled material containing sequences complementary
to those spotted on the microarray. This type of microarray,
was first developed at Stanford University (Schena et al.,
1995; Shalon et al., 1996). During years of study, quite
a few types of DNA microarrays have been developed
into promising tools for the genome-wide analysis of
transcripts (Roy et al., 2002). The application of DNA
microarray has extended to nutrigenomics, toxicology, and
food safety, in areas such as genetically engineered foods
and food-borne pathogens (Liu-Stratton et al., 2004).
Although the public has not displayed complacency
regarding the introduction of GM technology, large
expanses of cultivated land are still being used in
transgenic field trials resulting in market introduction
of GMOs and their derivatives, either as import or
export commodities. Hence, it is felt that apart from
the existing detection methods, a more efficient high-
throughput detection method is needed, to screen
GMOs being introduced to the market. In this study we
propose microarray technology as a prototype for high-
throughput detection of pure GMOs. The transgenic
tomato, potato, and rice in our laboratory were screened
for genes such as promoters, candidate genes, endogenous
control genes, reporter genes, selection marker genes,
and termination signals by a microarray chip. For proof-
of-concept we extended practicality to the detection of
genetic modifications in market-introduced GM maize and
cornflakes. Results clearly portray microarray as a potent
and reliable GMO detection system for pure samples,
including monocots and dicots.
MATERIALS AND METHODS
Type of materials
Transgenic and non-transgenic rice, potatos, and
tomatos developed in our laboratory were used. Samples
analyzed included seed and leaf materials in rice, fruit and
leaf materials of tomato, and tuber and leaf materials of
potato. Transgenic maize seeds (Bt11, T25 and NK603)
were kindly provided by the National Laboratories of
Foods and Drugs, Taiwan. Wild type maize and wheat
provided by Dr. Shih (Bureau of Food and Drug Analysis,
Department of Health Executive Yuan, Taipei, Taiwan)
served as a non-transgenic control. Cornflakes and wheat
samples obtained randomly in the market were a product
of California, USA.
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CHEN et al. — Microarray detection of GMOs
3
Raw material dilution
Raw material of transgenic and non-transgenic potato
tubers were sliced and mixed by weight in the proportion
of 1:20, 1:100, 1:1000, 1:10,000 and 1:50,000 for the
sensitivity assay of our system. Twenty gram of mixed
samples was homogenized for isolation of total genomic
DNA.
DNA isolation
Total genomic DNA was isolated by the CTAB (cetyl
trimethyl ammonium bromide) method. DNA from
maize seeds was extracted by the method described by
Majchrzyk (Majchrzyk, 2002). Homogeneous samples
(20 g) of each crop type (transgenic and non-transgenic)
were homogenized in a blender. DNA from potato tubers
were extracted as described by Wulff et al. (2002).
The quality and concentration of DNA was determined
spectrophotometrically at 260/280 nm.
Cloning and construction of candidate genes
The vector used for cDNA library construction was
pT7Blue perfectly blunt vector (Novagen, Darmstadt, Ger-
many). Inserts of DNA clones were amplified using the
PCR primers shown in Table 1 (BgVV. Berlin, Germany,
1998; Hupfer et al., 1998; Lipp et al., 1999; Matsuoka et
al., 2001). The PCR cocktail mixtures (100 μl) contained
plasmid (30 ng) or genomic DNA
template (300 ng), 10 ×
reaction buffer [200 mM Tris-HCl (pH 8.0), 100 mM KCl,
100 mM (NH
4
)
2
SO
4
, 20 mM MgSO
4
, 1% Triton X-100,
1 mg/ml nuclease-free bovine serum albumin (BSA)], 5
μl 100% DMSO, 4 μl 2.5 mM dNTP, 1 μl 100 pmole/μl
5’ primer, 1 μl 100 pmole /μl 3’ primer, and 1 μl Pfu DNA
polymerase (5 units/μl). Amplification was performed in
the PCR thermocycler (GeneAmp 2400, Perkin Elmer,
California, USA) and consisted of 35 cycles (94°C for 3
min; 95°C for 1 min,
72°C, 30 sec, and 72°C for 3 min).
Amplification was monitored by fractionating in a 1%
agarose gel, stained for visibility with ethidium bromide.
The purified PCR product was eluted using a Viogene kit
(Viogene, Taipei, Taiwan) and cloned into pT7Blue Per-
fectly Blunt vector, as instructed by the kit manual.
cDNA microarray preparation
The microarray methods were a modification of those
reported by Seki (Seki et al., 2002). The cDNA library
products were arrayed from 384-well microtiter plates
onto
poly-L-lysine-coated micro slide glass (GAPSII,
Corning, USA) using the PixSys4500 System gene tip
microarray stamping
machine (Cartesian Technologies,
USA).
About 0.5 μl of PCR products (100-500 ng/μl)
were pipetted from the 384-well microtiter plates. Five
nl per slide were deposited onto six slides, spaced 280
μm apart. The printed slides
were rehydrated in a beaker
with hot distilled water and snap
dried at 100°C for 5-10
sec. DNA was cross-linked on the slide using a UV cross-
linker (150-300 mJ). The slides were placed into a
slide
rack, which was in turn placed into a glass chamber.
The blocking solution, containing 25 ml of 0.2 M, pH
8.0 sodium borate, (Sigma, Missouri, USA), and 225
ml of 1-methyl-2-pyrrolidone (Sigma, Missouri, USA),
was poured into the glass
chamber. The slide racks were
plunged up and down five times,
shaken gently for 15 min,
transferred into a chamber with
boiling water, and allowed
to stand for 2 min. The slide racks were then transferred
into another chamber containing
95% ethanol
for 1 min,
and centrifuged
at 500 rpm for 5 min.
Nucleotide labeling
The procedure for labeling genomic DNA by Klenow
reaction and Cy3- or Cy5- nucleotides were a slight
modification of protocol described by Eisen and Brown
(Eisen and Brown, 1999) and TIGR. In this modified
protocol, 2 μg of genomic DNA was digested with Sau3
AI (average size is 500-1,000 bp for improving labeling
efficiency). Purification of the digested DNA (Qiagen PCR
purification kit, Qiagen, Valencia, CA) was performed by
adding 1/10 volume of 3 M CH
3
COONa (pH 5.2) and 2
volumes of ethnol. The DNA was precipitated at -70°C
for 0.5 h or overnight at -20°C, followed by centrifugation
at 15,000 g at 4°C. The pellet was dissolved in sterile
distilled water. A cocktail PCR mixture containing 1 μg of
purified DNA, oligo-dT, and DEPC-water was incubated
in a PCR machine at 70°C for 10 min and snap chilled
on ice. Superscript buffer 0.1 M DTT, 10 mM dNTPs,
superscript II RT (Life Technologies, Rockvile, USA)
were added immediately and mixed thoroughly before
incubation in PCR machine for 1 h at 42°C. The labeling
reaction was cleaned up as described by TIGR standard
operating procedure (SOP) (Anklam et al., 2002).
Microarray hybridization and scanning
The hybridization method was also a modification of
that reported by Seki (Seki et al., 2002). The probe sam-
ples were
placed onto the center of a slide, and a cover slip
was placed over the entire array surface to avoid bubble
formation. Four 5 μl drops of 3 X SSC were
placed on
separate points on the slide, which was placed in a humid
hybridization chamber to prevent dehydration of the probe
mixture
during hybridization. The slides were placed
in a
sealed hybridization cassette (Genetix, Boston, USA) and
submerged in a water bath maintained at 65°C, for 12-16 h.
After hybridization, the slides were removed and placed
in a
slide rack submerged in washing solution 1 (2 X SSC,
0.03% SDS)
with the array face of the slide tilted down so
that the
cover slip dropped off without damaging the array
surface.
The racks were then plunged
up and down three
times in washing solution 1 and transferred
to washing so-
lution 2 (1 X SSC) carefully to minimize contamination of
the second chamber because SDS can
interfere with slide
imaging. The slide chamber was rocked gently
for 2 min.
The slide racks were then transferred to washing solution
3
(0.05 X SSC), allowed to stand for 2 min, spun at 500 rpm
for
5-10 min, and dried.
Microarrays were scanned with a scanning laser micro-
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4
Botanical Studies, Vol. 47, 2006
Table 1. The primers designed for PCR amplification of DNA clones.
Template
Genes Primers
Size (bp) Tm (°C) Ref
Selection marker
pCAMBIA 2301
NPTII sense- 5’ TCCGGCCGCTTGGGTGGAGAG
470 63.6 This study
anti- 5’ CTGGCGCGAGCCCCTGATGCT
pCAMBIA1201
HPH sense- 5’ AGCTGCGCCGATGGTTTCTACAA
509 60.5 This study
anti- 5’ ATCGCCTCGCTCCAGTCAATG
p932A-GUSR
aadA sense- 5’ AAGCGGTGATCGCCGAAGTATCGAC 455 59.9 This study
anti- 5’ AAAGAGTTCCTCCGCCGCTGGA
pJD4401
PAT sense- 5’ GCGGTCTGCACCATCGTCAA
415 63.1 This study
anti- 5’ AGTTCCCGTGCTTGAAGCCG
Reporter gene
pBI221
GUS sense- 5’ CTGCGACGCTCACACCGATACC
441 59.5 This study
anti- 5’ TCACCGAAGTTCATGCCAGTCCAG
pMTC54
LUC sense- 5’ GAGAATAACATTTTGATAGGACCAC 484 50.8 This study
anti- 5’ GCATAGATTGATACCCCAAG
pCAMBIA1304
GFP sense- 5’ AAGGAGAAGAACTTTTCACT
541 51.9 This study
anti- 5’ TGATAATGATCAGCGAGTTG
Promter and Terminator
pCAMBIA1304
35Sp sense- 5’ CATGGAGTCAAAGATTCAAA
500 47.2 Lipp, 1999
anti- 5’ ATATAGAGGAAGGGTCTTC
PJD301
Nost sense- 5’ CGTTCAAACATTTGGCAATA
253 52.5 Lipp, 1999
anti- 5’ CCCGATCTAGTAACATAGAT
pCAMBIA1304
35St sense- 5’ AATTCGGGGGGATCTGGATT
201 50.4 This study
anti- 5’ CGATCGACAAGCTCGAGTTTAT
TetVp16
Ocst sense- 5’ GCTAGCTATATCATCAATTTAT
204 44.8 This study
anti- 5’ CCCATCTTGAAAGAAATATAG
pMTC40
tmlt ense- 5’ TATTAGGTTACGCCAGCCCT
240 44 This study
anti- 5’ TAACACGCACACTTACGATA
Control Gene
Rice genomic DNA
Actin sense- 5’ GACTACTACAAGGGCATCAG
318 42
*
anti- 5’ CACACCCACTCCAGATGCCT
Maize genomic DNA
ivr sense- 5’ CCGCTGTATCACAAGGGCTGGTACC 226 52 This study
anti- 5’ GGAGCCCGTGTAGAGCATGACGATC
Soybean genomic DNA LE sense- 5’ GCCCTCTACTCCACCCCCATCC
11 8 48
*
anti- 5’ GCCCATCTGCAAGCCTTTTTGTG
Tomato genomic DNA b-tubulin sense- 5’ CCCGGGCACACTTTGATCCCATTCG 530 50 This study
anti- 5’ CCCGGGCATTCTGTCTGGGTACTCTTC
GM Soybean / Maize gene
Transgenic maize CBH351 sense- 5’ CCTTCGCAAGACCCT TCCTCTATA 170 50 Matsuoka, 2001
genomic DNA
anti - 5’ GTAGCTGTCGGTGTAGTCCTCGT
Transgenic soybean CP4EPSPS sense- 5’ TGATGTGATATCTCCACTGACG
172 45
*
genomic DNA
anti- 5’ TGTATCCCTTGAGCCATGTTGT
Transgenic maize
T25 aense- 5’ GCCAGTTAGGCCAGTTACCCA
149 45 Matsuoka, 2001
genomic DNA
anti- 5’ TGAGCGAAACCCTATAAGAACCCT
Transgenic maize
Bt11 sense- 5’ CCATTTTTCAGCTAGGAAGTTC
11 0 42 Matsuoka, 2001
genomic DNA
anti- 5’ TCGTTGATGTTKGGGTTGTTGTCC
Transgenic maize
GA21 sense- 5’ ACGGTGGAAGAGTTCAATGTATG
270 42 Matsuoka, 2001
genomic DNA
anti- 5’ TCTCCTTGATGGGCTGCA
Transgenic maize
CryIAb sense- 5’ ACCATCAACAGCCGCTACAACGACC 184 50 Hupfer, 1998
genomic DNA
anti- 5’ TGGGGAACAGGCTCACGATGTCCAG
Foot note: 35Sp: promoter from the cauliflower mosaic virus; 35St: CaMV35S poly (A) signal; aadA: streptomycin-resistance;
Actin: rice actin gene; PAT: gene coding for a phosphinothricin acetyltransferase from Streptomyces hygroscopicus; Bt11: specific
gene of Bt11 (Novartis); β-tubulin: tomato β-tubulin gene; CBH351: specific gene for CBH351 (StarLink, AgrEvo); CryIAb:
delta-endotoxin from Bacillus thuringiensis subsp. Kurstaki; CP4EPSPS: 5-enolpyruvylshikimate-3-phosphate synthase from
Agrobacterium tumefaciens strain CP4; GA21: specific gene of GA21 (Monsanto); GFP: green fluorescent protein gene; GUS:
alpha-glucuronidas e gene; HPH: hygromycin phosphotransferase gene; ivr: maize invertase gene; LUC: luciferase gene; LE:
soybean legumin protein gene; NOSt: terminator of nopaline synthase gene from Agrobacterium tumefaciens; NPT II: neomycin
phosphotransferase gene; Ocst: octopine synthase terminator; tmlt: transcription terminator of a tumor morphology large gene
from Agrobacterium tumefaciens; T25: specific gene of T25 Libery (AgrEvo).
*: http://www.methodensammlung-lmbg.de/
pg_0005
CHEN et al. — Microarray detection of GMOs
5
scope (model
GenePix4000B; Axon Instruments, Union
City, USA). Separate images were acquired
for each fluor
at a resolution of 10 μm per pixel. In order to
normalize
the two channels with respect to signal intensity,
we ad-
justed photomultiplier and laser power settings so that
the
signal ratio of the β-tubulin genes (internal control) was
as close to 1.0 as possible.
We used Imagene, Version 5.0
(Gene Spring, Redwood City, CA) software for the micro-
array data analysis.
RESULTS
Set up of array analysis system
The set up of our microarray analysis system was
based on the studies of rice, tomato, and potato transgenic
plants, which were established by our laboratory.
Transgenic and non-transgenic rice (Oryza sativa, cultivar
TNG67) was arrayed for the detection of endogenous
genes such as invertase, LE, Actin, and β-tubulin, in
addition to genes such as the CaMV 35S promoter driven
mammalian protein gene containing nopaline synthase
from Agrobacterium, 35S terminator, and a hygromycin
phosphotransferase selection marker in transgenic rice,
developed in our laboratory. As is clearly evident in Figure
1 (A and B) none of the foreign genes of transgenic rice
were observed in the untransformed rice, which served
as the negative control (Figure 1B left). However, in
transgenic rice all the mentioned above component genes
of the expression cassette were detectable (Figure 1B
right) in addition to the endogenous genes. Non-specific
genes were not detected.
A similar experimental setup was used to discriminate
between non-transgenic tomato (Lycopersicon esculentum)
and transgenic tomato, the former serving as a negative
control. Non-GM tomato array slide revealed signals
of the endogenous genes such as invertase, legumin,
actin, and β-tubulin, but none of the foreign genes of
the expression cassette were observed (Figure 2B left).
However transgenic tomato harboring a reading frame
consisting of CaMV 35S promoter driven Arabidopsis
CBF1 transcription factor, nopaline synthase terminator,
35S terminator, nptII selection marker gene, and a gus
reporter gene were detectable, in addition to the genes
detected in non-transgenic tomato. No non-specific genes
were detected on this slide (Figure 2B right).
Transgenic potato (Solanum tuberosum L.) transformed
with a CaMV 35S driven phytase gene and containing
a nopaline synthase terminator, 35S terminator and a
kanamycin selection marker were arrayed along with
a non-transgenic potato, which served as a negative
control. As expected endogenous genes for invertase, LE,
Actin, and β-tubulin were detected in both crop types,
but the foreign gene components were observed only in
the transgenic potato lines (Figure 3B left). Non-specific
genes were not detected on this slide (Figure 3B middle).
For proof-of-concept purposes, we arrayed a market-
introduced commercial GM potato sample for the
above mentioned genes. As depicted in Figure 3B right,
in addition to the endogenous genes detected in non-
transgenic potato, we could also detect T-DNA harboring
genes such as 35S promoter, nopaline synthase terminator,
nptII selection marker, and one foreign gene from Bacillus
thuringiensis, CryIAb, in this sample. Similarly wild
type and market wheat samples were also arrayed for
GM detection, but no proof for genetic modification was
evident (Figure 4).
Figure 1. Non-transgenic and transgenic rice detected by mi-
croarray slide: Panel A, genetic construct from GM rice. Abbre-
viations are as follows. Ubi, maize Ubiqutin promoter, IFN-g,
human interferon-gamma gene. 35Sp, HPT, nos, and 35St are
as described in Table 1; Panel B left, non-transgenic rice (Oryza
sativa, cultivar TNG67) detected through the presence of ivr,
LE, Actin, and β-tublulin endogenous genes. Panel B right,
transgenic rice (Oryza sativa, cultivar TNG67) detected through
the presence of additional transgenic genes 35Sp, 35St, Nost,
and HPT; Panel C, the position of specific genes spotted on the
slide.
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6
Botanical Studies, Vol. 47, 2006
Figu re 3. Non-transgenic and transgenic potato detected by
microarray slide: Panel A, genetic construct of GM potato. The
abbreviation phytase (myo-inositol hexakisphosphate phospho-
hydrolase) is from Selenomonas ruminantium (Yanke et al.,
1998); Panel B left, non-transgenic potato (Solanum tuberosum
L.) was detected through the presence of ivr, LE, Actin, and
β-tublulin gene; Panel B middle, transgenic potato (Solanum
tuberosum L.) was detected through the presence of additional
transgenic genes 35Sp, 35S t, Nost and NPTII. Panel B right,
unknown potato sample detected through the presence of 35S
promoter, nopaline synthase terminator, kanamycin s election
marker, and one foreign gene from Bacillus thuringiensis, Cry
IAb; Panel C, the position of specific genes spotted on the slide.
Figure 2. Non-transgenic and transgenic tomato detected by
microarray slide: Panel A, The genetic construct of GM tomato.
The abbreviation CBF 1 indicates the C-repeat/de hydration
responsive element binding factor 1 from Arabidopsis; Panel
B left, non-transgenic tomato (Lycopersicon esculentum) was
detected through the presence of ivr, LE, Actin, and β-tublulin
gene. P anel B right, transgenic tomato (Lycopersicon escul-
entum) detected through the presence of additional transgenic
genes 35Sp, 35St, Nost and NPTII; Panel C, the position of spe-
cific genes spotted on the slide.
Microarray detection of GM and non-GM maize
seeds
In the present investigation, we validated cDNA
microarray as a detection tool for three varieties of
genetically modified maize (T25, Bt11 and NK603). Non-
transgenic maize (Zea mays) seeds served as a negative
control. An array of data depicted in Figure 5 shows
that, on the non-GM slide, signals related to endogenous
genes for invertase, LE, Actin, and β -tublulin were
detected, but not for the mechanistic genes of the T-DNA
commonly used in transformation events. However as
expected, these genes were detected on our array slide
and revealed the presence of CaMV35S promoter gene in
the events Bt11 and NK603, and the bar gene coding for
a phosphinothricin acetyltransferase from Streptomyces
hygroscopicus was detected in events T25 and Bt11. The
CaMV35S terminator was only observed in event T25
while the terminator of nopaline synthase (nos) gene was
observed by the events Bt11 and NK603. For the gene of
delta-endotoxin, Cry1Ab was detected in event Bt11, and
event NK603 was found to harbor the CP4EPSPS gene.
In order to prove the precision of the microarray system
in weeding out GMOs from a heterogeneous population,
GM maize seeds of the above mentioned transformation
events were mixed together in equal proportions. DNA
samples arrayed from this germplasm mixture revealed
exactly similar patterns as observed individually for each
transformation event. A signal for nptII was not observed
in the array blot, meaning that the event Mon810 was
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CHEN et al. — Microarray detection of GMOs
7
missed during sampling. Further, the fact that no false
genes were observed in the array blot proved beyond
doubt that the microarray is a reliable and accurate system
for detecting GMOs.
Microarray detection of GM and non-GM
cornflakes
Assessing the genetic modification in 3 GM maize
exemplifies cDNA microarray as an efficient detection
system for GMOs. However for proof-of-concept
purposes, a safety assessment measurement was conducted
in two types of branded commercial cornflakes introduced
to the Taiwan market and imported from USA. As
expected, we observed that all the endogenous genes used
were detected in the two brands of cornflakes. In addition,
gene components corresponding to various transformation
events were detected in these flakes as depicted in Figure
6. In the first cornflake brand, sample 1 (blot1), CaMV35S
promoter, nos terminator, bar gene, CP4EPSPS, Cry1Ab,
Bt11, and GA21gene were detected. This implies that
maize samples of Bt11 and GA21, probably containing
event NK603, might also have been slipped in as
derivatives. Similarly, in cornflake sample 2, the array
revealed the presence of CaMV 35S promoter, nos
terminator, CaMV 35S terminator, bar gene, CP4EPSPS
gene, Cry1Ab, Bt11, and T25 gene, implying that the
transformation events Bt11, T25, and NK603 were used.
Until now, totally 119 samples have been tested in this
system including GM and non-GM crops (Table 2). Only
one soybean sample showed a false result. That means the
mean accuracy percentage is 99%.
Sensitivity assay
To examine the limitation of our detection system, the
DNA samples were prepared after mixing transgenic and
non-transgenic potato tubers in respective proportion. The
Figure 4. Νοn-transgenic wheat and marketing wheat sample
detected by microarray slide: Panel A left, non- transgenic wheat
(Triticum aestivum L.) detected through the presence of ivr,
LE, Actin and β-tublulin gene. Panel A right, marketing wheat
sample detected through the same presence of ivr, LE, Actin,
and β-tublulin gene; Panel B, the position of specific genes
spotted on the slide.
Figure 5. Array data depicting the maize components detected
in the non-transgenic and transgenic maize seeds: Blot non-
GM: non-transgenic maize, Blot T25, Bt11, NK603: the genes
detected in the transgenic maize. Panel A right-corner, details of
specific genes spotted on the slide; Panel B, the construction of
T25, Bt11, NK603.
pg_0008
8
Botanical Studies, Vol. 47, 2006
compositions of the samples in Figure 7 were as follows:
5% GM potato tuber; 1% GM potato tuber; 0.1% GM
potato tuber; 0.01% GM potato tuber and 0.002% GM
potato tuber. DNA samples arrayed from these mixtures
correctly identified the foreign genes and endogenous
genes from 5% to 0.01% dilution samples. However, two
blurred spots (spots of 35S promoter and nptII gene) were
observed in a further dilution to 0.002% of GM potato
tuber, implying that the limitation of this detection system
ranged from 0.01 to 0.002% of GM crops in raw material.
DISCUSSION
Today, GM food is a topic of debate in the scientific
community and among the public and is a media focus.
Legal authentication by competitive authorities regarding
use of GM as food/feed has raised concerns among the
public due to the lack of strong evidence for the safety of
such foods on a long term basis. Though GMO regulations
over the release of such food products into the market
have been tightened, lack of universality has given rise
to skepticism among the public regarding the stringency
observed in different countries. Hence the public has
demanded a selection of GM-foods that is on a par with
non-GM foods. This has necessitated development of
an efficient and reliable GMO detection system that will
enable high-throughput screening to check intercontinental
Figu re 6. GM cornflake sam ples arrayed for the de tection
of maize components: Panel A, in two cornflake samples the
presence of specific endogenous and genetic modifications were
detected; Panel B, details of specific genes spotted on the slide.
Table 2. The GM and non-GM samples detected by this system.
Examples
No.
GMO
Non-GMO
FALSE
Tomato
18
2
16
0
Rice
16
3
13
0
Maize
25
5
20
0
Soybean
20
6
14
1
Potato
26
2
24
0
Wheat
14
0
14
0
Figure 7. Array sensitivity assay. Raw material of GM potato
tubers was mixed in the proportion of 5%, 1%, 0.1%, 0.01% and
0.002%. Endogenous internal control genes ivr, LE, Actin, and
b-tublulin genes and transgenes 35Sp, 35St, Nost, and NPTII
were detected clearly in the slides 5%, 1%, 0.1% and 0.01%.
Presence of transgenes 35Sp, and NPTII was blurred in the slide
0.002%.
pg_0009
CHEN et al. — Microarray detection of GMOs
9
gene flow and solve legal disputes arising from GMO
controversies. The present investigation is an attempt to
develop an efficient and reliable detection method for
GMOs exemplified by the use of cDNA microarray.
In the present investigation, GMO detection techniques,
exemplified by the use of a microarray detection system,
overcome limitations encountered in the existing detection
systems. The microarray detection system we designed
has advantages like accuracy, precision, reliability, and
reproducibility and is well suited to the high-throughput
detection of pure GMO samples. For proof-of-concept
purposes, the present study demonstrates the feasibility
of detecting mechanistic T-DNA genes in the transgenic
rice, tomato, and potato developed in our laboratory.
In addition we extended our arraying system for the
detection of a market-derived commercial transgenic
potato and wheat, with no changes observed in the quality
of the detection system. Due to its high accuracy, speed,
and precision qualities, microarray detection can be
effectively implemented for the qualitative detection of
GMOs in industries and regulatory laboratories to address
disputes pertaining to GMOs in future, where a large
number of samples have to be screened in a time-result
manner. The advantages of microarray detection include
screening for a large number of different GMOs within
a single experiment (Aarts et al., 2002). The feasibility
of a microarray detection system to weed out genetic
components in processed food and food products has been
proven in our lab with soybean and processed soybean
foods.
This microarray system also has been used for the
detection and identification of three GM maize lines (T25,
Bt11 and NK603), the transformation details of which
were known. These products were developed by USA-
based companies and have been marketed all around
the world. According to the results in Figure 4, it can
be assumed that microarray will meet internationally
prescribed standards for GMO detection.
Past strategies for introducing GMOs depended on
consumer requirements, but in the near future GMO
development will become more complex. Crossing of
approved GM varieties are considered to be novel GM
varieties. Due to the easier amplification of specific
sequences in the PCR product by universal primers (T7
and U19), the production of our GMO microarray is
accomplished.
The strength of any GMO detection assay lies in its
accuracy, reliability, and ease of use. Earlier DNA and
protein based assays have reportly been unable to achieve
100% accuracy in detecting the samples. When compared
with the traditional food-born pathogen detection methods,
the microarray method displayed a significantly higher
sensitivity. Recently, the report of using spotted DNA
microarrays labeled with oligonucleotide probes that were
complementary to four virulence loci (intimin, Shiga-
like toxins I and II, and hemolyxin A), E. coli O157:H7, a
food born pathogen was detected from a less than one-cell
equivalent of genomic DNA (1fg) (Call et al., 2001).
According to a 2002 proclamation of the Taiwan
Department of Health, all food products prepared from
raw material containing GM soybean and maize greater
than 5% are now subject to mandatory labeling prior to
public sale (http://www.doh.gov.tw/cht/index.aspx). The
European Union also introduced legislation stipulating the
mandatory labeling of food products with GMO content
exceeding 1% (Anonymous, 1998). In this study, we
also tested the quantitative sensitivity of our microarray
system. A serial dilution of GM potato tuber raw material
was used as a model for this assay (Figure 7). The
detection limits were in the range of 0.01-0.002% of the
mass fraction of GMOs. This result is sufficient for the
application of this system to the detection of genetically
modified material.
In our results, microarray detection assay was able to
achieve almost 100% accuracy, exemplified by its ability
to weed out GM maize components in three non-labeled
branded cornflake samples. Low template concentrations
can generate significant amounts of non-specific
amplification products in PCR. This makes interpretation
of the results more complicated and biased (Minunni et al.,
2000). Microarray analysis involves more than one target
in each reaction, enabling a crosscheck of the results in the
same reaction with other targets.
Due to the microarray methods high accuracy, speed,
and precision, it can be implemented for the qualitative
and quantitative detection of GMO in industry and in
regulatory laboratories. Though we have not tried to adapt
the microarray system to a quantitative detection analysis
of GMO, we feel this methodology can be highly useful if
used in combination with other effective methods, such as
quantitative PCR.
Acknowledgments. We thank Dr. Virginia Walbot for
providing pJD301 plasmid DNA and the Center for
the Application of Molecular Biology to International
Agriculture for providing the serial pCAMBIA vectors.
This work was supported by a grant from Academia Sinica
and a grant from the Department of Health of the Republic
of China.
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CHEN et al. — Microarray detection of GMOs
11
以 cDNA 微矩陣的方法建立大量篩檢基因改造食品 (GMOs)
的模式
陳姿利
1
 SANJAYA
2
 Venkatesh PRASAD
2
 李慶華
3
 林冠宏
4
 闕麗卿
5
 
詹明才
2
1
私立東方技術學院食品科技系
2
中央研究院生物農業研究所籌備處
3
私立中國文化大學生物技術研究所
4
私立中國文化大學園藝系
5
行政院½生署藥物食品檢驗局
  為了發展出一套有效且快速的基因改良食品檢驗系統,本實驗搜集了一般常見於基因改良作物的
外來 DNA 基因片段如花椰菜鑲嵌病毒 CaMV 35S 啟動子, NOSt 或 35S 終結子、 nptII 、 hph 或 pat
篩選基因、gus 或 gfp 標示基因,cp4 epsps、cry1 Ab 等己知的轉殖基因和 4 個植物的內標準基因 (如
invertase, legumin, β-tubulin, actin 基因) 等,打點於微型晶片上建立出一個大量篩檢基因改良食品的
cDNA 微矩陣系統。研究中先以本實驗室自行完成的轉殖水½、蕃茄及馬鈴薯做初步的檢測和系統敏感
度測試。結果顯示,外來基因 CaMV 35S 啟動子、 NOSt 、35S 終結子、 nptII 和 hph 等基因都能成功地
檢測出來,而相對應的野生種則只檢測出 4 個植物的內標準基因。為了更進一步了解此系統對市售樣品
的檢測效率,實驗中也測定 GM 馬鈴薯及 GM 玉米及玉米脆片食品和市售小麥種子,結果同樣可以明
½的偵測到外來 DNA 基因的存在。而在以馬鈴薯所做的敏感度測試中也顯示此系統至少可以偵測到原
料中只含有 0.01-0.002% 的 GM 馬鈴薯塊莖, 因此證明這個 cDNA 微矩陣系統可以發展成一個快速且
便於大量篩檢的檢測系統。
關鍵詞:cDNA 微矩陣法;基因轉殖物種 (GMO);基因轉殖馬鈴薯;基因轉殖玉米。
pg_0012