Biotecnología

 

Assessment of DNA extraction methods from various maize (Zea mays L.) tissues for environmental GMO monitoring in Mexico. Part I: detection by end-point PCR

 

Evaluación de métodos de extracción de ADN de varios tejidos de maíz (Zea mays L.) para el monitoreo ambiental de OGM en México. Parte I: detección por PCR punto final

 

Andrea SanJuan-Badillo¹, Amanda Galvez², Javier Plasencia¹, Maricarmen Quirasco²

 

¹ Department of Biochemistry. Faculty of Chemistry. National Autonomous University of Mexico. 04510. Mexico City, Mexico.

² Department of Food and Biotechnology. Faculty of Chemistry. National Autonomous University of Mexico. 04510. Mexico City, Mexico. (quirabma@unam.mx)

 

Received: January, 2013.
Approved: November, 2013.

 

Abstract

In Mexico, regulations for growing genetically modified (GM) maize (Zea mays L.) plants have been enforced in order to prevent gene flow to native landraces and wild relatives. Field surveys are necessary and Mexican government agencies have the mandate to perform them. Because of their specificity, PCR-based methods are suitable for field monitoring of GM organisms but it is necessary to assess their performance, since they are greatly influenced by the DNA preparation quality inherent to the extraction method. In this study, genomic DNA was extracted from various maize tissues (e.g. pollen, leaves, spikelets and grains) using five different commercial purification protocols. DNA quality was analyzed spectrophotometrically, by gel electrophoresis, and as a substrate for end-point PCR. Results showed that highly amplifiable DNA, rather than high extraction yields, is needed for a consistent analysis. Criteria to evaluate DNA purity, such as absorbance, do not necessarily reflect an adequate amplification capability, resulting in a non-reliable GM organism detection. In conclusion, silica DNA-binding membranes yielded the most suitable DNA preparations for end-point PCR analyses of different GM maize tissues.

Key words: PCR inhibition, GM maize, variability of DNA yield, DNA quantification, GMO detection.

 

Resumen

En México se han implementado reglamentaciones para el cultivo de plantas de maíz (Zea mays L.) genéticamente modificadas (GM) con el fin de evitar el flujo de genes a razas nativas y especies silvestres. Los estudios de campo son necesarios y las agencias del gobierno mexicano tienen el mandato para realizarlos. Debido a su especificidad, los métodos basados en la PCR son adecuados para la vigilancia en campo de organismos GM, pero es necesario evaluar su desempeño, ya que están muy influenciados por la calidad de la preparación de ADN inherente al método de extracción. En este estudio se usaron cinco protocolos comerciales diferentes para extraer y purificar ADN genómico de varios tejidos de maíz (por ejemplo, polen, hojas, espiguillas y granos). La calidad del ADN se analizó por espectrofotometría, por electroforesis en gel, y como un sustrato para PCR punto final. Los resultados mostraron que se necesita ADN altamente amplificable, en lugar de altos rendimientos de extracción, para un análisis consistente. Los criterios para evaluar la pureza del ADN, como la absorbancia, no necesariamente reflejan una capacidad de amplificación adecuada, lo que resulta en una detección no fiable de un organismo GM. En conclusión, las membranas de sílica de unión de ADN dieron las preparaciones de ADN más adecuadas para los análisis de PCR punto final de diferentes tejidos de maíz GM.

Palabras clave: Inhibición de PCR, maíz GM, variabilidad de rendimiento de ADN, cuantificación de ADN, detección de OGM.

 

Introduction

Mexico is the center of origin and diversification of maize (Zea mays L.), as evidenced by a wide diversity of open-pollinated landraces and the presence of maize wild relatives (CONABIO, 2009), but Mexico imports annually 8×10⁶ t of yellow maize from the USA (SIAP, 2011) for food, feed and processing. Transgenic maize hybrids with different agronomic properties, biotechnologically developed, occupy about 85% of the 32×10⁶ ha of maize grown in the USA (GMO Compass, 2010; USDA, 2012). Maize imported from that country is not identified or labeled as genetically modified (GM), and escapes and misuse of this grain is an environmental risk, though there are some controversial evidences of transgene introgression from GMO events to native maize (Ortiz-Garcia et al., 2005; Piñeyro-Nelson et al., 2009). Transgenic maize is grown under strict regulations implemented by the Mexican government, only for experimental and pilot field trials in order to prevent gene flow from biotech hybrids to open-pollinated landraces (CIBIOGEM, 2012a).

In order to pursue this issue, government agencies must perform constant monitoring with verified molecular analytical methods, mainly based on PCR, to trace these events in the field; such activities are implemented by the Mexican Interministerial Commission on Genetically Modified Organisms (CIBIOGEM, 2012b), in accordance to regulations in Mexico (Ley de Bioseguridad de organismos genéticamente modificados, 2005; Reglamento de la Ley de Bioseguridad de organismos genéticamente modificados, 2009; CIBIOGEM, 2012b). Sampling in the field requires analyzing different tissues for environmental risk assessments: fresh leaves, dry leaves, pollen, seeds, stalks and grains. Adequate sampling protocols must be developed in conjunction with appropriate and validated methods of extraction, amplification and detection of the possible exogenous GM sequences. The availability of validated methods, certified reference materials, as well as all reagents and extraction kits is crucial for implementing suitable routine methodologies. The objective of this study was to test various protocols for DNA extraction from different maize tissues and to assess yield and quality for further qualitative PCR analyses, in order to provide rational recommendations to government agencies' laboratories to implement the appropriate molecular techniques for field sample processing and analysis.

 

MATERIALS AND METHODS

Plant material

Seeds from an open-pollinated white maize landrace were obtained from a local grower in Chalco, Mexico (August 2005). Embryos were excised manually from grains. Seedlings (two-week old) were grown from seeds in a greenhouse at the Faculty of Chemistry, UNAM to collect leaf tissue. Adult plants were grown for 10 to 12 weeks to collect leaf samples and spikelets. Leaf samples from seedlings and adult plants were dried in an incubator at 37 °C. Tassels were collected from a commercial maize field in Los Reyes, Estado de Mexico (October 2005), as well as dehiscent pollen. GM reference material and certified maize grains from three commercial events were provided by Genetic ID NA, Inc. (Fairfield, IA, USA). Grains from the MON810, Bt11 and NK603 GM hybrid varieties were ground separately in a high-speed blender to obtain a particle size <1 mm, before analysis. Seeds from the GM hybrids germinated under controlled conditions and leaves from two-week seedlings and mature plants were collected.

Genomic DNA extraction

Genomic DNA was extracted from different tissues and samples using five commercial methods available in Mexico: 1) Fast DNA^® kit (Qbiogene. Carlsbad, CA, USA); 2) DNeasy plant kit (Qiagen, Inc. Germantown, MD, USA); 3) DNAzol^® reagent (Invitrogen Co. Carlsbad, CA, USA); 4) Easy-DNA™ (Invitrogen, ibid); and 5) Fast DNA extraction kit (Genetic ID NA, Inc. Fairfield, IA, USA). Manufacturer instructions were followed, and they are described briefly. Method 1): 200 mg grounded tissue sample was mixed with CLS-VF and PPS solutions; it was incubated on ice and then shaken in the FastPrep FP120 homogenizer (30 s). After centrifugation (14 000 x g), a 600-µL aliquot was mixed with 600 µL of the binding matrix. The mixture was incubated 5 min at room temperature, centrifuged at 14 000 x g, the supernatant was discarded and the pellet was resuspended in SEWS-M solution for washing. After centrifugation, the supernatant was discarded and the DNA was dissolved in DES solution. Method 2): a 100 mg tissue sample was frozen with liquid nitrogen and ground in a mortar. The ground tissue was transferred to a clean tube and AP1 buffer and RNase A were added, followed by 10-min incubation at 65 °C. AP2 buffer was added and the mixture was incubated for 5 min on ice and then passed through a Mini Spin QI column by centrifugation. The filtrate was mixed with AP3/E buffer and the mixture was passed through a DNeasy mini spin column, the column was washed twice with AW buffer and finally the DNA was eluted with AE buffer. Method 3): a 50 mg tissue sample was frozen with liquid nitrogen and ground in an Eppendorf tube with a glass pestle. DNAzol was added to the tube and mixed gently, the tube was centrifuged at high speed and the supernatant was mixed with isopropanol (−20 °C). The tube was incubated 3 min at room temperature and then centrifuged 10 min at 14 000 x g. The pellet was washed with 70% ethanol, dissolved in 10 mM NaOH and neutralized with 1M Tris, pH 8.0. Method 4): a 50 mg tissue sample was frozen with liquid nitrogen and ground. Solution A was added and the mixture was vortexed, it was incubated 10 min at 65 °C and solution B was added and vortexed again. Chloroform was added to the mixture and then centrifuged to separate phases, the upper phase was mixed with absolute ethanol (—20 °C) and incubated on ice 30 min. After centrifugation, genomic DNA was washed with 80% ethanol, dissolved in TE buffer and treated with RNase A. Method 5): a 1 g sample was mixed with Fast-Lyse buffer, Proteinase K and RNase A, and incubated 30 min at 37 °C. Chloroform was added, the mixture was vortexed and then centrifuged 15 min. A 0.9 mL aliquot of the upper phase was taken and mixed with 0.9 mL of Fast-Bind solution. The mixture was centrifuged and the supernatant was passed through a silica membrane DNA binding column, which was washed with Fast-Bind solution and later with 75% ethanol. Excess of ethanol was eliminated by centrifugation and DNA was eluted with TE buffer.

Genomic DNA was extracted from pollen by grinding it with liquid nitrogen and then following the described protocol for each method. These DNA extraction methods are suitable for isolation of genomic DNA from plants and represent distinct purification approaches. Method 1) is based on lysis by shearing in a chaotropic buffer and isolation of DNA by binding to a silica matrix and washed. In methods 2) and 5), the tissue is chemically lysed, DNA is bound selectively onto a silica-based membrane and several washings are performed before DNA elution. In method 3), a guanidine-detergent lysing solution is used and DNA is precipitated with isopropanol. In method 4), the tissues are incubated in a solution that contains lytic enzymes, followed by a chloroform cleanup and DNA precipitation with ethanol.

Spectrophotometric and electrophoretic analyses of genomic DNA

A UV scan from 200 to 380 nm was performed for all DNA extractions (Shimadzu UV160U UV/visible spectrophotometer). Genomic DNA concentration was estimated considering that a value of A₂₆₀ of 1.00 corresponds to 50 ng µL^-1 of double-stranded DNA (Sambrook and Russell, 2001). The absorbance at 320 nm was considered for background estimation. DNA yield (ng mg^-1) was calculated considering the total DNA extracted and the amount of tissue employed for each method. Genomic DNA samples (approximately 125 ng per lane) were resolved by horizontal gel electrophoresis in 0.8% agarose gels in 1X Tris-Acetate-EDTA buffer. Gels were stained with ethidium bromide (0.5 mg mL^-1) and photographed under UV light (Sambrook and Russell, 2001).

End-point PCR amplification

PCR reactions were performed using 100 ng of genomic DNA. Validated PCR primers specific for the amplification of Z. mays invertase gene (ivrl), 35S promoter and nos terminator (Table 1) were used at 0.3 µM, plus 0.2 mM dNTP, 2.5 mM MgCl₂ and 0.02 U µL^-1 Platinum Taq DNA polymerase (Invitrogen, Co. Carlsbad, CA, USA) per reaction (20 µL). Cycle conditions were the same for the three amplicons: 1 cycle at 94 °C for 5 min, followed by 35 cycles of 45 s at each one of 94, 56 and 72 °C, and a final cycle at 72 °C for 7 min. The reactions were run in a GenAmp^® PCR system 9700 Thermocycler (Applied Biosystems Inc. Foster City, CA, USA). PCR products were resolved by horizontal electrophoresis in 2% agarose gels, and 50-bp or 100-bp DNA ladder (Promega Co. Madison, WI, USA) were used accordingly as size markers. Gels were stained with ethidium bromide (0.5 mg mL^-1) and photographed under UV light. Non-template controls were included in each PCR run. All amplicons obtained were cloned in a pGEM vector and sequenced to verify identity.

 

RESULTS AND DISCUSSION

Spectrophotometric assessment of DNA yield and purity

There was a high degree of heterogeneity among the different extraction methods evaluated as DNA yield. Methods 1), 2) and 3) yielded DNA in the same value range (Figure 1), except for some of the tissues extracted with method 2), specifically grain and dry plant leaf. In contrast, method 4) yielded DNA one order of magnitude higher than the other methods with values from 472 ng mg^-1 in grains to 4120 ng mg^-1 in dry leaf (Figure 1). Even for a single method, there are noticeable differences depending on the type of plant tissue tested. Although in a kernel about 50% of the extracted DNA comes from the endosperm and the other 50% from the embryo (Trifa and Zhang, 2004), low DNA yields from whole grains were typically obtained because of the high starch content in the endosperm. Given the high DNA content in embryos, after their manual dissection, DNA was obtained in the best case with a 20-fold enrichment with method 2). DNA from embryos was used as an experimental ideal sample not proposed to be performed as a tissue for a routine analysis procedure, but that could be useful for special individual plant analyses. In general when leaves were dried, a two-to-four-fold increase in DNA yield was obtained with methods 1) and 3) with respect to the fresh tissue. In contrast, method 2) yielded the same or a lower amount of DNA in dry tissue when compared with the fresh counterpart. Spikelets and pollen were considered as tissues to be analyzed due to their potential application in gene flow and cross fertilization studies to define isolation distances for transgenic maize plots (Sanvido et al., 2008). Pollen grains were not easily lysed using any of the manufacturer protocols, so a grinding step with liquid nitrogen was performed before DNA extraction. In this manner, satisfactory DNA yields were obtained from a low amount of tissue (Figure 1).

A fifth DNA extraction method (method 5) was also included in this study in view of the good quality DNA it rendered in a study by Quirasco et al. (2008). DNA from grains and fresh leaves rendered low yields similar to the ones obtained with method 2), being 21 and 35 ng DNA mg^-1 tissue. Although UV spectrophotometry (A₂₆₀) is widely used for nucleic acids quantification, it has shortcomings: it measures double-stranded DNA but if the sample carries RNA, single-stranded DNA or other compounds, such as phenols, and absorbance increases accordingly. Both, DNA degradation and denaturation contribute to this hyperchromic effect. Degraded DNA may yield single-stranded fragments and upon denaturation, strand separation occurs (Demeke and Jenkins, 2010; Gryson, 2010). Reliable, absolute quantification of DNA by UV spectrophotometry is difficult to achieve unless a pure double-stranded DNA sample is dissolved and evenly distributed in solution. Other methods to quantify DNA rely in intercalating agents, such as SYBR Green and PicoGreen. These fluorescent dyes bind preferentially to double-stranded DNA yielding, a very sensitive method. However, SYBR Green methods may overestimate DNA concentrations by 15 to 50% (Nielsen et al., 2008).

DNA extracted is intended for further molecular analyses requiring high purity in order to obtain reliable and consistent results. For PCR analysis, DNA suitability is usually assessed measuring absorbance at 230, 260 and 280 nm (Table 2). The A₂₆₀/A₂₈₀ ratio indicates the DNA preparation purity regarding to the presence of contaminant proteins, and although the values obtained in most DNA preparations seemed to be adequate (values between 1.8 and 2.0) (Sambrook and Russell, 2001; Gryson, 2010), it is not an absolute criterion. A further quality indicator for DNA extracts can be obtained from the ratio of A₂₆₀/A₂₃₀, it is recommended to be ≥1.7, which means that the A₂₆₀ would lead to an accurate DNA quantification. A value below 1.7 is associated with the presence of carbohydrate and phenolic compounds that may interfere with PCR (Corbisier et al., 2007; Demeke et al., 2009; Gryson, 2010). For most of the DNA preparations, the A₂₃₀ value was well above 2.0, so the corresponding ratio could not be calculated (Table 2). Results showed that only a few of the samples obtained meet the mentioned standard value, and that purity assessed by absorbance was not necessarily related to amplification capability by a given DNA template, as shown below.

Some buffer components present in the DNA extraction kits might contribute to the absorbance. Substances like guanidine-HCl, EDTA or phenol could be carried over the process and absorb at 230 or 280 nm. Other compounds that might be in the plant such as aromatics, humic acids, carbohydrates, and peptides absorb at 230 nm as well (Sambrook and Russell, 2001; Demeke and Jenkins, 2010). A scan throughout 200 and 320 nm was useful for detecting contaminations or carryover of reagents from the extraction procedures. UV scan profiles from DNA extracted with methods 1) and 3) showed outstanding out-of-range values at wavelengths below 260 nm. Moreover, no inflection point was observed, probably indicating an important carryover of the chaotropic salt used in the extraction procedure.

Electrophoretic evaluation of DNA integrity

Because the most common sources to obtain DNA for GMO analyses are grains and fresh leaves, these preparations were further analyzed for DNA integrity in 0.8 % agarose gels. Tissue source also affected DNA integrity, as illustrated for methods 2) , 4) and 5) which showed a smear in DNA from leaf, consistent with DNA fragmentation; however, DNA molecular size was big enough to obtain good amplification, as shown further on by end-point PCR. In the case of method 1) for leaf DNA, any band could be observed. Due to the fact that A₂₆₀ was used to estimate DNA concentration in order to load 125 ng per lane, it is clear that DNA amount was overestimated because of the presence of contaminants that absorbed at that wavelength. In contrast, DNA obtained from dry grain was observed as a distinct high-molecular weight band, in particular the ones obtained with methods 1), 4) and 5). Method 3) yielded DNA with a similar electrophoretic pattern for both tissues, as a faint high-molecular weight band with a discrete smear underneath and an intense high-mobility smear. Quirasco et al. (2004) applied method 5) to detect and quantify the cry9C transgene in highly processed corn products such as baked and fried tortillas. Despite the extreme pH and temperature treatments, that lead to DNA degradation and make difficult the PCR analysis (Gryson, 2010), down to 0.1% (w/w) GMO was detected in most products.

DNA as substrate for PCR amplification

A wide array of methods have been developed and adapted to extract and purify DNA for various agricultural diagnostic applications. Purification methods are based on different principles that range from DNA precipitation to anion-exchange techniques and selective adsorption of DNA. Demeke and Jenkins (2010) point out 18 DNA extraction methods and kits; some of them have variations or are coupled, e.g., the CTAB (hexadecyltrimethylammonium bromide) DNA extraction procedure followed by a DNA purifcation kit. An essential test for PCR-based analyses is to determine if the extracted DNA is a good substrate for DNA polymerase amplification, as plant tissues contain compounds that inhibit this reaction. The biochemical composition of each tissue is quite different, representing a whole array of compounds that hinders the attainment a DNA preparation suitable for molecular analysis. Whereas the whole maize grain contains high amounts of polysaccharides, green leaf tissues contain phenolic and polyphenolic compounds that might also interfere with the DNA polymerase activity (Gryson, 2010). Neutral polysaccharides such as 2% starch and 4% dextran, do not inhibit the PCR amplification. In contrast, the acidic polysaccharides alginic acid and dextran sulfate, as well as glucuronic acid, inhibit amplification, even at concentrations below 0.02% (Holden et al., 2003). Plant phenolic compounds co-purified with DNA may inhibit the PCR amplification by binding to the DNA polymerase or to the DNA itself (Wilson, 1997; Schrader et al., 2012). To assess this, the amplification of a 225-bp fragment of a Z. mays endogenous gene, invertase (ivr1), was performed using the DNA extractions obtained before as templates. Methods 2), 3) and 4) were chosen because they represent different methodological approaches, and they showed the extremes regarding DNA yield. According to results, not all preparations obtained with methods 3) and 4) yielded amplifiable DNA, in contrast to the amplification achieved from all DNA samples extracted with method 2) (Figure 2). These results were consistently obtained in the replicated samples and reactions, suggesting the presence of an inhibitor of the PCR in DNA preparations coming from methods 3) and 4). It is noteworthy that for method 4) the highest DNA yields were obtained (Figure 1), as well as an acceptable UV spectrum and DNA integrity for preparations coming from fresh tissues. However, DNA amplification of the endogenous gene could not be achieved from DNA preparations for all tissues tested. These results might be explained by a non-accurate DNA estimation that led to an underload in the PCR mix, whereas other substances that absorb at 260 nm are included in the reaction (Holden et al., 2003). Particularly the DNA extracted from dry tissues with methods 3) and 4) (Figure 2, lanes DPL and DSL) did not yield any amplicon due to the fact that inhibitors might be more concentrated than in the corresponding fresh tissues. There was a brownish color in dry tissues, which might be caused by phenolic compounds and polymers such as lignin. These contaminants might explain amplification inhibition.

In Table 2, dry tissue values for A₂₆₀/A₂₈₀ are approximately 1.4, indicating either a high protein concentration or the presence of phenolic compounds in relation to DNA amounts. As for embryo tissue, contaminants not easily detectable spectrophotometrically might be present. The results obtained were similar to other reports concluding that methods yielding high DNA concentration might contain impurities that hinder proper amplification, as compared to those yielding a lower amount of DNA compromising yield in order to produce a more purified preparation (Terry et al., 2002; Corbisier et al., 2007). Altogether, these results reflect the diverse composition of the various maize tissues employed, and that not all extraction methods could yield acceptable DNA preparations across the board.

The limitations of some of the methods tested were confirmed when DNA was extracted with the same five methods from grain and plant leaf from three transgenic events (NK603, MON810 and Bt11) and tested as substrate for end-point PCR for the amplification of invertase gene, as before. For all these reactions, a non-template control was included and no amplicon was obtained (data not shown). DNA prepared with methods 1), 2) and 5) served as an adequate template for PCR amplification, generating consistently a 225-bp amplicon regardless of tissue or maize genotype (Figure 3). Amplification from DNA prepared with method 1) reflected the fact that DNA from leaves is usually more fragmented than DNA from grains, as the band intensity of the amplicon from the three leaf samples is clearly lower than that obtained from grains. Besides, an inaccurate DNA estimation by A₂₆₀ could have led to an overestimation resulting in a lower DNA load in the PCR. DNA prepared with methods 3) and 4) was a poor template for PCR with very inconsistent amplification of the endogenous gene.

The same DNA samples were also used in end-point PCR to detect the CaMV 35S promoter and the Agrobacterium tumefaciens nos terminator sequences, two common elements in genetically engineered plants. Again, the DNA prepared by methods 2) and 5) yielded a consistent 123-bp amplicon corresponding to the CaMV 35S promoter, regardless of the tissue or maize genotype (Figure 4). DNA prepared with methods 1), 3) and 4) also yielded the amplicon, but in an inconsistent manner, as faint bands were observed for some preparations. A similar behavior was observed for DNA preparations when the 118-bp amplicon from the nos terminator was synthesized, due to the fact that DNA extracted with methods 3) and 4) was the less adequate for a reliable amplification (data not shown). The identity of all amplicons presented in the previous results was verified by sequencing the respective clones.

As for GM detection, these results show that DNA quality (purity and integrity) is essential for an accurate analysis. In a similar study, six DNA extraction and purifications methods were tested to analyze Roundup Ready^® soybean. The authors found that the DNA extraction and purification method has a profound effect on the recovery and quality of DNA. Actually, the method that produce the best preparation for qRT-PCR was DNA extracted with CTAB and further purified with a fast spin column technology (Demeke et al., 2009). A study comparing eight extraction methods to isolate genomic DNA from several medicinal plants found that DNA preparations varied widely in yield and purity. Because the DNA was used in PCR-based sequencing reactions the authors suggest to expand the A₂₆₀/A₂₈₀ range to values between 1.3 and 2.3 where DNA samples might be amplifiable (Llongeras et al., 2013). Such criteria are useful for this type of studies as a broad range of tissues were tested and each one of them presents certain difficulties to yield high-quality genomic DNA.

As this investigation was prompted by the Ministry of Environment and Natural Resources of Mexico to develop techniques for high-throughput screening of GMO events, a post-PCR hybridization assay could be implemented for confirmation. For example, detection and confirmation of transgenes in soybean is achieved by the use of an aequorin-oligonucleotide conjugate that allows the bioluminometric hybridization of CaMV 35S promoter, nos terminator and lectin DNA sequences in microtiter wells (Glynou et al., 2004). In the second part of this investigation, DNA preparations obtained with the same commercial kits are evaluated for their performance in transgene quantification by real-time PCR.

 

CONCLUSIONS

Although ratios of A₂₆₀/₂₈₀ and A₂₆₀/₂₃₀ are considered to assess DNA purity, PCR inhibitors might be present in certain tissues and are not detected by spectrophotometry, thus leading to false negative results. The methods that involve the use of silica membrane columns and washing buffers showed to be an efficient combination for PCR inhibitors removal; besides, they are the most time-saving and practical, especially when a large number of samples must be processed. This study shows that adequate DNA preparations for PCR amplification could be obtained from several maize tissues and even from plant debris. This comparison of methods provides useful information for the Ministry of Environment analytical laboratories, so they can be implemented along with appropriate field sampling protocols in order to track the presence in the field of GM maize events.

 

ACKNOWLEDGEMENTS

This project was financed by CONACYT-SEMARNAT (grant 2004-C01-266). We thank M. Nájera-Martínez (UNAM, Faculty of Chemistry) for her technical assistance.

 

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