Agric. Chem. Biotechnol. 47(4), 185-188 (2004)
Article
Qualitative PCR Method for Detection of Genetically Modified Maize Lines NK603 and TC1507
Seong-Hun Lee1, Yong-Hwan Park, Jin-Kug Kim1,2, Keun-Woo Park2 and Young-Mi Kim*
Gene Expression Team, National Institute of Agricultural Biotechnology, Suwon 441-100, Korea
1Division of Inspection, Experiment Research Institute of National Agricultural Products Quality Management Service, Seoul 150-043, Korea
2Division of Bioscience and Technology, College of Life and Environmental Sciences, Korea University, Seoul, Korea Received November 18, 2004; Accepted December 17, 2004
Qualitative analytical method was developed using polymerase chain reaction (PCR) for detection of new lines of genetically modified (GM) maize, NK603 and TC1507. Two specific primer pairs were designed for qualitative PCR. To confirm specificity of designed primers, PCR was performed on genomic DNA extracted from new and other GM maizes, and cereal crops. Single PCR product was obtained from each designed primer pairs. For sensitivity determination of designed primers, test samples including NK603 and TC1507 GM lines were prepared at 0~10% levels and analyzed by PCR.
Limit of detection (LOD) was 0.01% for both NK603 and TC1507 GM maizes. The results showed that the developed PCR method was useful for specific detection of NK603 and TC1507 lines of GM maize.
Key words: PCR, genetically modified (GM) maize, limit of detection (LOD)
In recent years, several agricultural biotechnological companies worldwide have developed a number of genetically modified (GM) crops through the recombinant DNA technology, and many countries have authorized the commercialization of genetically modified organisms (GMOs) as food and feed. Accordingly, with the increasing importation of GM grains, GMO labeling system has become a requirement. The purpose of the labeling system is to inform the consumers of the presence of GMOs in the product, thus providing the consumers with the option to select their preferred products. Many countries have established labeling systems based on their own criteria, with thresholds for unintentional mixing of GM crops defined as 0.9, 5, and 3% in the EU, Japan, and Korea, respectively.1-3)
Maize (Zea mays L.) is the worlds third leading cereal crop, following wheat and rice, and also the most commercialized GM crop. The cultivated area of GM maize is 23% of total maize area worldwide.4) However, Korea imports almost its entire maize requirement; in 2002, Korea imported 99.3% of its maize demand. With the implementation of Korea’s GMO labeling regulation in 2002, the development of detection methods was required for GMO monitoring. The polymerase chain reaction (PCR) is one of the most widely used methods for detecting the presence of GMOs.5)
Qualitative PCR analysis detects whether the test sample contains GMO, which kinds of GM lines, and whether or not the GMO is approved. 6-9) Up to the present, eight lines of GM maize have been analyzed through qualitative PCR methods.10-12) Recently, two new lines of GM maize, NK603 and TC1507, have been approved for commercialization and importation to Korea. NK603 GM maize line is a glyphosate- tolerant maize that contains 5-enolpyruvylshikimate-3- phosphate synthase (EPSPS) gene from Agrobacterium tumefaciens CP4 strain.13) TC1507 line is an insect-resistant and glufosinate-tolerant maize that contains two transgenic genes, Cry1Fa2 and PAT (phosphinothricin-N-acetyltransferase).
The Cry1Fa2 gene was isolated from Bacillus thuringiensis (Bt) var. aizawai, a delta-endotoxin. The PAT gene was isolated from Streptomyces viridochromogenes. It allows the use of phosphinothricin herbicides including glufosinate ammonium, which inhibits glutamine synthetase of the plant.
The PAT enzyme catalyzes the acetylation of phosphinothricin and detoxifies it into an inactive compound.14)
In this study, two kinds of construct-specific primer pairs were designed to detect the two new GM maize lines, NK603 and TC1507. The specificity and sensitivity of the new qualitative PCR method were confirmed.
Materials and Methods
Maize and other crops. Seeds of two new GM maize lines, NK603 (DKC57-40) and TC1507 (Herculex I), were provided by Monsanto Company (St. Louis, MO, U.S.A) and Dow AgroSciences Company (Indianapolis, IN, U.S.A), respectively. Conventional non-GM (hybrid control) maize
*Corresponding author
Phone: +82-31-299-1641, Fax: +82-31-299-1642 E-mail: [email protected]
Abbreviations: P-ract 1, rice actin 1 promoter; E35S pro, enhanced CaMV 35S promoter; NOS, Nopaline synthase; Ubi ZM pro, ubiquitin Zea mays promoter; ORF25, open reading frame 25
186 Seong-Hun Lee et al.
was provided by Dow AgroSciences Company. Five lines of GM maize, Bt11, MON810, GA21, T25, and Event176, were used to study the specificity of the designed primer pairs. The other crops used were rice (Oryza sativa), ‘Ilpoom’ variety, and barley (Hordeum vulgare), ‘Saessal’ variety.
DNA extraction. The maize, rice, and barley seeds were ground into fine powder using an electric mill (Fritsch pulverisette 14, City, Germany). Using DNeasy Plant Maxi kit (Qiagen, Germany) following the manufacturer’s instructions with a minor modification, genomic DNA was extracted from 1-g ground samples of each crop. The quantity and the quality of the extracted DNA were monitored by UV spectrophotometer DU530 (Beckmann Coulter Inc., USA) and agarose gel electrophoresis.
Oligonucleotide primers. To detect a GMO, it is necessary to develop the specific primer pair for GM line. All oligonucleotide primer pairs were designed using Primer Express software version 1.5 (Applied Biosystems, USA).
The primers were synthesized and purified on PAGE columns by Bioneer Company (Daejeon, Korea). Figure 1 and Table 1
show the positions and the sequences of primer pairs.
SSIIb primer pair detected the maize starch synthase IIb (zSSII) gene as an endogenous reference control. The P35S and tNOS primer pairs detected the widely used genes in GM crops, CaMV 35S promotor gene and nopaline synthase, respectively. These primer pairs were synthesized based on the DNA sequences previously reported by Kuribara et al.10)
TC-F195/R445 primers were designed to amplify a region containing both maize ubiquitin (MubG1) promoter and Cry1F gene based on the deposited sequences (GeneBank Accession No. U29159 and No. M73254) for detection of the TC1507 maize line (Table 1). NK-F163/R393 primers were designed to detect NK603 line based on the junction region between heat shock protein 70 gene (Gene Bank Accession No. X03697) and chloroplast transit peptide2 gene (Gene Bank Accession No. X06613).
Qualitative PCR. PCR was performed to confirm the specificity of the designed primers in genomic DNAs extracted from GM maize lines including two new lines NK603 and TC1507, rice, and barley. To determine the Fig. 1. Schematic diagrams of the designed PCR primer pairs for detection of two GM maize lines, (i) NK603 and (ii) TC1507.
Closed arrows indicate the specific regions for detection of NK603 and TC1507 GM maizes. Open arrows indicate the universal regions of p35S and tNOS for GM maize.
Table 1. List of primer pairs for qualitative PCR
Target Name Sequence (5' 3') Specificity Length (bp)
Endogenous
zSSIIb SSIIb 1-5' CTC CCA ATC CTT TGA CAT CTG C zSSIIb 151
SSIIb 1-3' TCG ATT TCT CTC TTG GTG ACA GG zSSIIb
Universal
CaMV35S P35S 1-5' ATT GAT GTG ATA TCT CCA CTG ACG T p35S 101
P35S 1-3' CCT CTC CAA ATG AAA TGA ACT TCC T p35S
tNOS Tnos 2-5' GTC TTG CGA TGA TTA TCA TAT AAT TTC TG tNOS 151
Tnos 2-3' CGC TAT ATT TTG TTT TCT ATC GCG T tNOS
Construct-specific
NK603 NK-F163 CCT CCT GAT GGT ATC TAG TAT CTA CCA ACT hsp-70 231
NK-R393 GAG AGA TTG GAG ATA AGA GAT GGG TTC epsps
TC1507 TC-F195 CTG CCT TCA TAC GCT ATT TAT TTG C ubiquitin 251
TC-R445 GGA ACA AAC TCA GAC AAC AGG AAA C cry1F
Qualitative Detection Methods of Genetically Modified Maize 187
sensitivity of the qualitative PCR method, DNA mixture was prepared for each of the two GM maize lines and the non-GM maize line at the following levels: 0, 0.01, 0.05, 0.1, 0.5, 1.0, 3.0, 5.0, and 10.0% (v/v).
All the amplifications were carried out in the GeneAmp PCR System 9700 (Applied Biosystems, USA) at 25µl reaction volume with 50 ng genomic DNA, 2.5µl 10ÜPCR buffer, 0.2 mM dNTP, 1.5 mM MgCl2, 0.5µM each primer, and 1.25 units AmpliTaqGold polymerase (ABI). The PCR conditions were as follows: 94oC for 10 min, and 40 cycles of 94oC for 30 sec, 60oC for 30 sec, 72oC for 30sec, and 72oC for 7 min.
Sequence Analysis. All PCR products were subcloned into TOPO TA cloning vectors (Invitrogen, USA). DNA sequencing was performed by CoreBioSystems (Seoul, Korea).
Results and Discussion
Specificity of the primer pairs designed for GM maize.
The primer pairs SSIIb, CaMV35S, and tNOS were used for the primary screening of GM maize.10,12,15) SSIIb primer pair was designed to differentiate maize from other grasses such as rice, barley, and wheat. CaMV35S and tNOS are universal primer pairs that can distinguish GM maizes from conventional non-GM maizes.
Although the primer pairs can screen out GMOs from a test sample of corn grains, primer pairs cannot detect the lines of GM maize mixed in the test sample. The construct introduced into NK603 maize line contained both CaMV35S promoter and NOS terminator genes; while TC1507 maize line contained only CaMV35S promoter gene (Fig. 1). The results
confirmed that CaMV35S and tNOS primer pairs could not screen all GM lines, suggesting the need for a qualitative detection of GMOs to differentiate GMOs from non-GMOs and identify GM lines.
In this study, two kinds of specific primer pairs were designed to screen and identify two GM maize lines with increased specificity of PCR (Fig. 1). Each primer pair successfully differentiated a single line from others, and each primer pair was specific to NK603 and TC1507 (Fig. 2).
Amplified fragments of 231 and 251 bp were observed when NK603 and TC1507 DNA were amplified as templates, respectively, whereas no amplifications were observed from the non-GM maize, the other five lines of GM maize, rice, and barley. These results demonstrated that the two primer pairs were highly specific for NK603 and TC1507 maize lines in PCR analysis. This specificity is basically attributable to the specific primer pairs designed to amplify the region containing both a trait gene and a regulatory gene, such as a promoter. The specificity results suggested that the developed qualitative PCR detection methods for the GM maize lines, NK603 and TC1507, were suitable for practical use in the monitoring and identification of GMO.
Sensitivity of the primer pairs designed for GM maize.
Sensitivity tests were performed to determine the limits of detection (LODs) of the new qualitative PCR method. The test DNA samples were prepared into nine mixing levels of 0, 0.01, 0.05, 0.1, 0.5, 1.0, 3.0, 5.0, and 10.0% (v/v) for each of the two GM maize lines.
The LODs for both NK603 and TC1507 GM maize were 0.01% (Fig. 3). This sensitivity level was similar to the qualitative detection levels of other GM maize lines, GM soybean, and GM potatoes.9, 16, 17) Using the lowest threshold value, which is 0.9% of that of EU, the results of sensitivity test demonstrated that the developed qualitative PCR method can meet the requirements for monitoring GMO labeling systems worldwide.
Acknowledgments. We are very grateful to the Monsanto Company for providing the seeds of NK603 GM maize Fig. 2. Specificity of the designed primer pairs for two GM
maize lines. PCR products were electrophoresed on 2% aga- rose gel. Arrowheads indicate the expected PCR amplification products. The primer pairs for detection of zSSIIb (A), p35S (B), tNOS (C), NK603 (D), and TC1507 (E) GM maizes were used. Template DNAs for each lane were as follows: Lanes 1- 11; No template control, Non-GM maize, MON810, Bt11, Event176, T25, GA21, NK603, TC1507, rice, and barley, respectively. M: 100 bp size ladder.
Fig. 3. Sensitivity of the designed primer pairs for two GM maize lines. PCR products were electrophoresed on 2% aga- rose gel. Arrowheads indicate the expected PCR amplification products. The primer pairs for detection of NK603 (A) and TC1507 (B) GM maizes were used. Template mixing DNAs for each lane were as follows: Lane1-11; 0, 0.01, 0.05, 0.1, 0.5, 1.0, 3.0, 5.0, and 10.0%, respectively. M: 100bp size ladder.
188 Seong-Hun Lee et al.
(DKC57-40), and to the Dow AgroSciences Company for TC1507 GM maize (HerculexI I) and non-GM maize.
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