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

2

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.

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-

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/

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.

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

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.

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%.

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。