Detection of genetically modified organisms (GMOs) by PCR: a brief review of methodologies available

Trends in Food Science & Technology 9 (1999) 380±388

Detection of genetically modi®ed organisms (GMOs) by PCR: a brief review of methodologies available E. Gachet,* G.G. Martin,* F. Vigneau* and G. Meyery *

Euro®ns Scienti®c S.A., Rue Pierre Adolphe Bobierre, B.P.42301, F - 44323 Nantes Cedex 3, France y Hanse Analytik GmbH, Fahrenheitstraûe 1, D-28359 Bremen, Germany The discovery of the molecules that bear the genetic information of any living organisms among which DNA (DeoxyriboNucleic Acid) plays a central role has been a revolution for life sciences. Taking the advantage of the universality of the genetic code, researchers have succeeded to associate DNA sequences coming from di€erent organisms using molecular biology techniques and to integrate foreign DNA within plants. These genetically-modi®ed organisms (the so called GMOs) have the ability to synthesise some additional proteins which confer new properties on them. Improving the protection of agricultural crops is one of the sought advantages by the gene transfer. According to the European regulation, the new foodstu€s made from genetically modi®ed soya or maize, must be labelled. The control of the labelling of such foodstu€s is based on the detection of the foreign DNA sequences born by the genetically-modi®ed organisms. One of the analytical methods used for enforcement of this regulation is the Polymerase Chain Reaction (PCR) method. The principle of

Review the PCR method is to multiply speci®c sequences of DNA, making them detectable. This highly sensitive method o€ers the advantage of detecting DNA molecules which are more thermostable than proteins. # 1999 Published by Elsevier Science Ltd. All rights reserved. As soon as human beings settled, they started to grow plants and breed animals. Step by step, they arti®cially created new species by selecting some types of plants and animals (criteria were yield, weight, strength, etc.). However, up to a few years ago, human beings were unable to control directly the biological process of this creation which is based on a random mix of genetic information through sexual reproduction. Though human beings have improved their techniques to control sexual reproduction, they have not yet acted directly at the core of the biological mechanism. They did not modify the genetic information that living organisms harbour. Life sciences have undergone enormous progress since the discovery that molecules bear genetic information. These molecules are called nucleic acids, among which DNA (deoxyribonucleic acid) plays a central role. The knowledge of the structure and the biochemistry of DNA, and mechanisms of DNA synthesis (polymerization, ligation, cutting) has allowed biologists to act directly on DNA sequences. Researchers have taken advantage of this easy manipulation of DNA and of the genetic code to associate DNA sequences coming from di€erent organisms. They have succeeded in building new DNA molecules by recombining di€erent DNA sequences with molecular biology techniques (Fig. 1). DNA is like a magnetic tapeÐit can be cut, moved and reinserted to create new or di€erent information. Thanks to molecular biology, researchers have succeeded in integrating foreign DNA within, for example, plant genomic DNA, but also in mouse or rat DNA. Genetically modi®ed organisms, abbreviated as GMOs, are the fruit of this research.

Advantages of GMOs

The chronology of gene expression is as follows. The gene is transcribed into its messenger ribonucleic acid (or mRNA). Then, the mRNA is translated into a protein. Thus, one protein corresponds to a speci®c gene. Introducing a foreign gene in a plant confers this on plant the ability to synthesize a foreign protein.

0924-2244/99/$ - see front matter Copyright # 1999 Published by Elsevier Science Ltd. All rights reserved. PII: S 0 92 4 - 2 24 4 ( 9 9 ) 0 0 00 2 - 3

E. Gachet et al./Trends in Food Science & Technology 9 (1999) 380±388

Based on the idea that any genetic information from any source can be expressed in any organism, genetic engineering has, for example, looked at improving the protection of agricultural crops. Other sought advantages include shortening the delay to obtain a new variety, improving the yield and quality of crops, producing

high value-added molecules (like pharmaceuticals or vitamins or biopolymers for industry), and improving the nutritional quality of plants (Tables 1 and 2). The ®rst genetically engineered fruit sold on the market was the well-known FlavrSavr tomato, which was designed to soften more slowly. A slower softening

Fig. 1. Di€erent steps for the creation of a GM corn containing an endotoxin gene.

Table 1. GMOs on sale in 1997 Crops

Property

Country

Company

Corn

Insect resistence

USA, Canada, Japan, EU

Soy bean

Herbicide tolerance Male sterility Herbicide tolerance

Tomato

Ripening slower

USA, EU, Switzerland USA USA, Canada, Japan, EU, Argentina, Switzerland USA, GB

Ciba-Geigy, Monsanto, Mycogen, Sandoz, Northrup King de Kalb, AgrEvo, Plant Genetic Systems Plant Genetic Systems AgrEvo, Monsanto

Potato Chicory

Insect resistance Male sterility and herbicide tolerance Virus resistance Virus resistance Virus resistance Male sterility Herbicide tolerance High level of lauric acid Composition of oil Insect resistance Herbicide tolerance Herbicide tolerance

Squash Melon Papaya Rape seed

Cotton Tobacco

381

USA, Canada, Japan Europe USA USA USA EU Japan, Canada, USA Canada USA USA, Mexico, Australia, Japan USA Europe

Agritope, Calgene, DNA Plant Technology, Monsanto, Zeneca Monsanto Bejo Zaden BV Asgrow, Upjohn Cornell U. Plant Genetic Systems AgrEvo, Monsanto, Plant Genetic Systems Calgene Calgene Monsanto Calgene/RhoÃne-Poulenc, Du Pont, Monsanto SEITA

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Table 2. GMOs under development in laboratories or undergoing ®eld testing Quality

Property researched

New GMOs

Agronomical

Herbicide tolerance

Wheat, sun¯ower, beetroot, lettuce, tomato, potato Sun¯ower, beetroot, tomato, rice, melon, squash, cucumber, tobacco Strawberry Banana Spinach, lettuce Potato Corn Soy bean Rice Strawberry Banana Broccoli Tobacco Cotton Poplar tree

Virus or fungus resistance

Nutritional

Pharmaceutical

Industrial

Production of an antifreeze protein Slower ripening Low amount of nitrates Richer in starch Lower level of saturated fats in oil High level of amino acids Not producing an allergen Higher levels of a natural anti-cancer agent Producing a vaccine against hepatitis B Producing anti-cancer and antioxidant agents Synthesizing haemoglobin Coloured ®bres Less ®bres

process means that the tomatoes can stay on the vine for longer which makes them tastier. Furthermore, tomatoes with elevated levels of an antioxidant, lycopene, may protect against cancer. Currently, the most widely inserted genes in GMOs confer resistance to worms, insects or to a herbicide. One of the aims has been to create a plant that resists any chemical protection used by farmers; for example soy bean or corn that are tolerant to herbicides like Round Up1. Round Up1 is a non-selective herbicide which acts by entering the plant and inhibiting an enzyme necessary for building aromatic amino acids. The lack of these amino acids kills the plant. Herbicide-resistant corn varieties designed to increase yields are already cultivated and a corn that has its own inbuilt insecticide is already sown and harvested in the United States. In addition, a lot of GMOs are under development. For example, genetically-modi®ed corn altered to make a healthier cooking oil by reducing its saturated fat content is on the way. In the case of strawberries, adding an antifreeze protein from the winter ¯ounder ®sh to help them grow in cold climates is under development, as are nutritionally enhanced strawberries with increased levels of a natural anti-cancer agent, ellagic acid. In the same way, for broccoli, a combination of natural anti-cancer and antioxidant agents could make this cruciferous vegetable essential eating as it would prevent or slow down the aging of cells that form living organisms. Soon, potatoes richer in starch will be used to produce low-fat chips and crisps that one might ®nd in the stores in 5 years' time. A higher starch level leads to a lower fat content because the potatoes cannot absorb as much fat when fried. Another fruit has been an issue of scienti®c research: the banana. Scientists are investigating whether genetically-modi®ed bananas could produce a vaccine against hepatitis B. Bananas

could also be made tastier if they can be induced to ripen more slowly. Two other plants are being engineered: the ®rst is a type of rice that would no longer produce an allergen factor, and the second is a lettuce that would have a lower amount of nitrate. Among GMOs, there are also a rape seed resistant to fungi or to herbicides, and GM chicory, papaya and squash. The genetically-modi®ed plants also concern nonfood crops like cotton with coloured ®bres or tobacco. Even the poplar tree is going to be genetically engineered to improve raw material (cellulose) for making paper.

Development of the market for GMOs

At the present time, genetically-engineered plants have been approved by several countries: the US, Canada, The European Union (EU), Switzerland, Australia, Argentina, Brazil and Japan. In the US, 20 genetically engineered products were approved in 1997 and 25 others have been submitted for approval. It is expected that in the next decade, 48 agricultural crops will be genetically modi®ed. In the EU, 12 varieties of GMOs have been approved to be sold on the European market. Nine other GMOs are going to be approved soon. Currently, the two most cultivated GMOs are corn and soya. In North America, genetically-modi®ed crops represented a small area of farmland in 1996, as only 1.5 million ha were covered with GMOs. As early as 1997, the cultivated area of genetically-modi®ed crops rose to 12.5 million ha (Fig. 2). This year, one-third of soy beans planted in North America were genetically-modi®ed, representing a two-fold increase over the previous year. There is a similar trend for corn. A signi®cant fraction of US corn, the staple constituent of biscuits, cakes and cookies, is already modi®ed. A total of 8.3

E. Gachet et al./Trends in Food Science & Technology 9 (1999) 380±388

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Fig. 2. The increase in the cultivated area of GMO crops in North America.

million ha of genetically modi®ed corn (20% of the total cultivated area) were planted in the US this year, up by 10% over 1997. It is forecast that the cultivated area of genetically-engineered crops will be multiplied more than three-fold by the year 2000, and that more than 66 million ha will contain transgenic plants by 2005 in North America. Some estimates suggest that in 10 years' more than 75% of major crops are going to be genetically engineered. Soy bean is a strategic crop for the food industry as well as for international trade. Indeed, soy beans are used today in 60% of all processed foodsÐ from margarine to chocolate, ice-cream, convenience and baby foods. In addition, North America produces the majority of the world's soy beans, sending up to 40% of the crop to Europe, an estimated 30% of which is genetically modi®ed.

Laws controlling the marketing of GMOs in the USA and the EU

By now, many countries have introduced legislation regulating the approval and release of GMOs. In the US, three independent authorities are involved in the regulation of the release of genetically engineered plants and their use as foodstu€s: APHIS (Animal and Plant Health Inspection Service), FDA (Food and Drug Administration) and EPA (Environmental Protection Agency). The APHIS controls movement between states, importation and culture assays of GMOs that might induce diseases in plants or be, by themselves, a disease. The FDA makes rules for additives and new foodstu€s, except meat, and products coming from poultry farming. The FDA also makes rules for animal medicines. The FDA is also concerned with the labelling of food. Currently, this administration considers that the composition of a food consisting of or derived from genetically modi®ed plants does not di€er signi®cantly from its conventional counterpart. Thus, the labelling of the genetically modi®ed organisms is not mandatory in the US.

By comparison the novel food regulations in the EU will require the labelling of GMO products if they can be distinguished from respective conventional products by scienti®c and analytical methods [1]. So, since September 1998, labelling has been made mandatory for foodstu€s containing ingredients derived from genetically modi®ed corn (the so-called `Bt-Maize' from Novartis) or soy bean (the so-called `RR-Soy' from Monsanto). As the genetically-modi®ed organisms bear supplementary genetic information in comparison with the conventional plants, the purpose of the analytical methods used for enforcement is to target these foreign DNA sequences. The control and detection of such foodstu€s will be possible thanks to the Polymerase Chain Reaction (PCR) method in addition to the conventional protein detection tests using antibodies like the ELISA-test (Enzyme-linked immunosorbent assay).

Detection of GMOs by the PCR method Principle of the PCR method as applied to GMO testing

The principle of this method is to amplify speci®c sequences of DNA thanks to a pair of short DNA sequences that ¯ank the region to be ampli®ed (these are known as primers). The PCR method is based on the molecular structure of DNA. The two strands that form the DNA molecule have a helical structure. Both of them are linked by periodic hydrogen bonds between the nucleotides (the elementary blocks of the sequence). Genetic information is like an alphabet based on four letters (the nucleotides, also named bases, are A, T, C, G). Each nucleotide binds to only one of three other nucleotides (A to T and C to G, and conversely T to A and G to C). The two strands are thus linked: a strand binds to its complementary strand. However, both strands of the double-stranded DNA can be separated from each other, under the action of a speci®c enzyme by breaking the links inside the region where the new strand of DNA will be polymerized. This

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is the ®rst step of DNA replication in vivo. When DNA has been separated into individual strands, single-stranded DNA is cut by a speci®c enzyme. Then, the polymerization of DNA can start. DNA polymerization may be divided into two steps. At ®rst, an enzyme called DNA polymerase binds to the end of the cut strand. Then, DNA polymerase synthesizes the new complementary strand. Each strand serves as a template for synthesizing the complementary strand. There is a polarity in extension of the new strand. As a matter of fact, the DNA polymerase links a new nucleotide to the 30 end of the extending strand, each strand having one end called 50 and the other end called 30 . Finally, two identical molecules of DNA are synthesized from one DNA molecule. The two daughter molecules are also identical to the mother molecule. The PCR mimics those three natural steps (Fig. 3). (1) The single-stranded DNA molecules are obtained by heating the DNA solution to a temperature of 94 C (enough to break the hydrogen bonds between the strands): this is the denaturing step. (2) By decreasing the temperature to around 55 C, a single-stranded DNA fragment can match its complementary single strand. Two short and arti®cially-synthesized DNA sequences (around 20±30 nucleotides), named primers, that ¯ank the region of interest to be ampli®ed bind to or hybridize to opposite strands. This is the annealing step. Template DNA, primers, DNA polymerase and bases are submitted to the reaction at the beginning of the whole PCR. (3) Finally, during the polymerization

step or extension step, the DNA polymerase synthesizes the new strand. The polymerization step that is carried out at around 72 C succeeds thanks to a DNA polymerase functional at high temperatures. Early on, this thermo-stable enzyme was extracted from the bacteria strain Thermus aquaticus, which lives in hot water springs. The standard PCR consists of about 20±50 cycles of denaturation (single-stranded DNA form), primer annealing and DNA synthesis. The target DNA sequence can be ampli®ed many times with these several rounds of denaturating and polymerization of DNA. Such a repetitive series of cycle results in the exponential accumulation of a speci®c fragment whose termini are de®ned by the 50 ends of the primers. Because the primer extension products synthesized in one cycle can serve as templates in the next, the number of target DNA copies approximately doubles at every cycle. Thus, 20 cycles of PCR yields about a million-fold ampli®cation. This highly sensitive method allows rapid in-vitro ampli®cation of small amounts of DNA fragments. Under ideal conditions, less than 10 copies of a speci®c DNA sequence are sucient to be ampli®ed by PCR into a readable signal. This amount is then enough for the DNA to be analysed. The kinetics of PCR depends on the concentration of the DNA and on its quality (Fig. 4). The more concentrated the DNA, the more quickly the targeted sequence is ampli®ed. Conversely, the more DNA is fragmented, the less targeted sequence is ampli®ed quickly because the starting of the exponential ampli®-

Fig. 3. Scheme of the Polymerase Chain Reaction (PCR).

E. Gachet et al./Trends in Food Science & Technology 9 (1999) 380±388

cation is delayed. However, this problem of DNA fragmentation has an e€ect only when highly processed food is analysed. Frequently, the degree of fragmentation is checked before starting the PCR. After the PCR, it must be determined if a fragment with the expected length has been ampli®ed. The PCR products are subjected to the standard electrophoresis technique. If there is any doubt about fragment identity, the ampli®ed fragment can be checked more precisely to determine that the PCR product is de®nitely the sequence of interest. Several methods may be used to do this: . a special gel electrophoresis technique which separates the DNA fragments according to their length as well as according to their base composition, e.g.

385

using bisbenzimide-PEG as a ligand of a speci®c DNA sequence [2] . the DNA ampli®ed may be digested by an enzyme that cuts the expected fragment into subfragments whose size is known . the Southern blotting technique (the PCR products are tested with a probeÐthe target DNA synthesized arti®cially) . the fragment ampli®ed may be sequenced to check the order of base pairs of the DNA sequence. The principle of the mainly used standard electrophoresis technique is the following: once the DNA target is ampli®ed, the DNA solution is loaded on an agarose gel. As they are negatively charged in the running bu€er, DNA molecules will move through the gel under the in¯uence

Fig. 4. Kinetics of PCR.

Fig. 5. Example of standard electrophoresis of DNA: demonstration of the presence as well as the size of ampli®ed DNA molecules (amplicons).

386

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of an electric ®eld. The segregation of the di€erent fragments of DNA is based on their size (Fig. 5). The smaller ones are seen at the bottom of the gel. After the electrophoresis is done, DNA is stained using a ¯uorescent molecule that binds to DNA and visualized under a UV light.

Di€erent methods of DNA extraction used for GMO analyses

Before the ampli®cation, DNA must be extracted from samples to analyse. It is a puri®cation step in which other types of molecules (proteins, lipids and polysaccharides) are thrown away. A lot of protocols allow this, but there are two main principles of extraction. Either, the crude solution of DNA is cleaned from impurities by di€erent agents, or DNA molecules are ®xed on a resin with high anity for them before being eluted. The CTAB-method [3] (CTAB means Cethyltrimethyl-ammonium-bromide) is based on the ®rst principle, and the Wizard-extraction method [4] is based on the second principle. The CTAB-method is divided in ®ve steps: . solubilization of DNA by addition of the CTABbu€er made with the detergent CTAB (at a ®nal concentration of 20 g/l) . denaturation of a large amount of proteins contained in the sample . ®rst precipitation of DNA with the second CTAB solution (the concentration of CTAB is lower: 5 g/l) . second degradation of residual proteins . second precipitation of puri®ed DNA with alcohol addition. The Wizard-extraction method is divided in three steps: . solubilization of DNA by addition of the extraction bu€er and proteinase K (an enzyme that

degrades proteins non-speci®cally as it cuts the covalent bond that links amino acids) . the crude DNA solution goes through a column containing the resin . DNA puri®ed is eluted from the resin. The CTAB-method is the basis for an ocial German Method [5] and the Wizard-extraction method has become the ocial Swiss Method [6].

Choice of di€erent target DNA sequences

Taking account of genetic building of GMOs, several speci®c sequences can be detected. These sequences can be classi®ed in three categories. . First category: sequences that regulate expression of transgenes. They are often formed by the sequence of the promoter P35S (a promoter extracted from the cauli¯ower mosaic virus which regulates the transcription of this virus) and by the sequence of the terminator Tnos (Tnos that regulates the end of the transcription of the nopaline synthase, comes from the bacteria strain Agrobacterium tumefaciens). Both of them are widely used to build the di€erent GMOs. . Second category: the genes that are used as genetic markers to be followed the transgene through the whole process of genetic building. They may, for example, confer antibiotic resistance to GMOs. . Third category: the target genes (transgenes) the most widely introduced in plants are genes conferring herbicide tolerance to plants (e.g. the gene that codes a phosphinothricine acetyltransferase) and a gene conferring an insecticide tolerance to plants (the gene codes a -endotoxin, cryIA(b)). In the frame of the European market, the plants currently approved and sold contain these genes.

Table 3. Some primers used for DNA ampli®cation Test Screening ``Roundup Ready'' soybean

Soya ``Maximizer'' maize Maize

Set of primers used

Target DNA sequence

Size of the PCR product

35S-1/35S-2 NOS-1/NOS-3 RR01/RR02 RR04/RR05 GMO5/GMO9 GMO7/GMO8 p35s-f2/petu-r1 GM01/GM02 GM03/GM04 CRYIA1/CRYIA2 CRYIA3/CRYIA4 Cry03/Cry04 ZEIN1/ZEIN2 ZEIN3/ZEIN4 ivrI-F/ivrI-R

35S promoter NOS terminator EPSPS gene EPSPS gene EPSPS gene EPSPS gene EPSPS gene Lectin gene Lectin gene Endotoxine gene Endotoxine gene Endotoxine gene Azain gene Azain gene Invertase gene

195bp 180bp 508bp 179bp 447bp 169bp 171bp 413bp 117bp 420bp 189bp 211bp 485bp 277bp 226bp

E. Gachet et al./Trends in Food Science & Technology 9 (1999) 380±388

Examples of primers

The detection of P35S or Tnos sequences may be carried out with two sets of standard primers. The primers 35S-1 and 35S-2 are the forward and reverse primers for an ampli®cation of a 195bp-fragment of P35S (Table 3). NOS-1 and NOS-3 are the forward and reverse primers for an ampli®cation of a 180bp-fragment of Tnos (Table 3). The detection of both sequences forms the screening method [7]. The detection of the gene enabling neomycin (an antibiotic) resistance may be carried out with the primers Tn5-1 and Tn5-2 [8] (Table 3). The detection of a speci®c gene that confers a herbicide resistance (the gene encodes the enzyme 3Enolpyruvyl-Shikimate-5-Phosphate-Synthase [EPSPS], i.e. a phosphinothricine acetyltransferase) may be carried out with the pair of primers RR01 and RR02 [4], or with GM05 and GM09 [9] (Table 3). These primers give rise, respectively, to a 508bp-amplicon and a 447bpamplicon. P35s-f2 and petu-r1 [10] amplify a 171bpfragment which contains the arti®cial link between the 35S promoter and a part of a gene from petunia (Table 3). The fragment is typical for Monsanto's Roundup Ready resistant soy. The detection of the gene that codes cryIA(b) may be carried out with the pair of primers CRYIA1 and CRYA2 [11] or Cry03 and Cry04 [12] (Table 3). These primers give rise, respectively, to a 420bp-amplicon and to a 211bp-amplicon.

Methodology for the analysis of a sample

To monitor the process of detecting DNA and the ampli®cation of sequences, several controls are necessary. There are positive controls using primers which will amplify a fragment that is in any case contained in the plant under investigation. They are designed for checking the quality of DNA preparation and the appropriateness of the general chemical parameters for DNA ampli®cation. The tests must give a signal after the ampli®cation and staining steps. A failure in the detection means that either there are inhibiting factors or there is not enough DNA in the DNA solution or the parameters used for the PCR are not right. For the analysis of a foodstu€ made from corn, the primers may be ZEIN1 and ZEIN2 [11] or ivrI-F and ivrI-R [12] (Table 3). The aim of these two sets is to detect, respectively, the gene coding azain or the gene coding invertase (corn-speci®c genes), in transgenic as well as in conventional corn. The control for foodstu€ derived from soy bean may be the pair of primers GM01 and GM02 [13] (Table 3). This set ampli®es a 413bp-fragment of a soy-speci®c gene that codes lectin. These primers give rise to a 413bp-amplicon. This product is detectable in conventional soya and in transgenic soya. Another positive control must be carried out to check that the parameters of the PCR (choice of primers, annealing temperature, time for each step of PCR, number of cycles for DNA ampli®cation) chosen for the

387

speci®c reaction are suitable. A plasmid bearing a speci®c sequence of GMOs (P35S, Tnos or an other speci®c sequence of the genetic modi®cation) may be used as a control DNA. In order to perform this test, the DNA sample can be divided in two halves. The control DNA is then added to one of the two halves. PCR products are expected at least with the trial containing the control DNA. If no PCR products are generated at the end of the PCR, it means that the PCR parameters chosen are not right for the speci®c reaction or that inhibiting factors might still be present. The negative control is designed to check that there is no contamination by modi®ed DNA in the laboratory. This trial does not contain any DNA. If a signal is produced it means that the procedures preventing contamination of the materials and solutions used for DNA extraction and ampli®cation have failed. It is also necessary to be careful about false positive or negative results. The detection of the P35S sequence alone is not proof that the foodstu€ is made from a GMO. As a matter of fact, a conventional plant contaminated by the cauli¯ower mosaic virus (P35S is originated from this virus) will also give a signal with the primers 35S-1 and 35S-2. Another explanation that may be given is that the primers can hybridize to non-speci®c sequences. In this case, the amplicon yielded does not correspond to the fragment of the targeted sequence. To prevent the misinterpretation of the results, sets of primers targeting other speci®c sequences from the genetically engineered organism must be used. DNA may not be visualized with a ®rst set of primers if DNA is highly damaged. This problem may be resolved by applying the nested-PCR technique. When the ®rst round of DNA ampli®cation has failed to produce a visible amount of amplicon because DNA is too fragmented, a second PCR is carried out using the outcome of the ®rst PCR as starting material with two other primers that can match to the DNA sequence within the region targeted the ®rst time. In the case of a successful assay, a smaller fragment will be produced. The following supplementary primers may be used (Table 3): . GM03/GM04 [13] (conventional and genetically modi®ed soy bean) yields a 118bp-amplicon . RR04/RR05 [4] or GM07/GM08 [9] (genetically modi®ed soy bean) yields a 179bp-amplicon or a 169bp-amplicon . ZEIN3/ZEIN4 [11] (conventional and genetically modi®ed corn) yields a 277bp-amplicon . CRYIA3/CRYIA4 [11] (genetically modi®ed corn) yields a 189bp-amplicon.

What is the bene®t of detecting DNA instead of proteins?

Nucleic acids are not considered as an interesting class of compounds in food chemistry. Indeed, the

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nucleic acids present in food have no nutritional value and there is no direct relationship between DNA and food quality. But whereas proteins are thermo-sensitive molecules, nucleic acids are very thermo-stable molecules. Upon processing, food proteins are no longer detectable or detectable with diculty because they are degraded. Conversely, nucleic acids are only slightly damaged by heat treatment. In addition, the PCR method is more sensitive than conventional protein detection tests. By comparison, the ELISA-test, based on using antibodies against speci®c proteins, a technique widely used in pharmaceutical tests, may be around 100 times less sensitive than the PCR method [13]. Furthermore, antibody tests such as ELISA are much more laborious and time consuming to develop and to validate than nucleic acid tests. So, thanks to this powerful and precise tool, a speci®c nucleic acid can be detected whatever the foodstu€ analysed, even in mixtures where the GMO ingredient is present in low concentrations. Another argument may justify the choice of DNA rather than protein detection. That is, whereas certain proteins may be expressed only in speci®c parts of the plant, like leaves, beans, pollen or stems, the whole genetic information is present everywhere in the plant, because the same genes are present in each cell of the plant. Thus, a broad range of products can be analysed by the PCR technique. Among the foodstu€s made from corn, there are products like corn germ, corn ¯our, corn pasta product, starch ¯our, whole corn, corn ¯akes, popcorn, corn chips, cakes, cookies, baked products and, if not in any case, even sugars derived from corn starch which can be analysed for a genetic modi®cation. The PCR technique is as well applied to a lot of matrices made from soya like for example soy bean, soy cream (liquid or lyophilized), soy milk (liquid or lyophilized), soy meal, tofu, meat products, soy ¯akes, lecithin, soy protein concentrate and, in several cases, depending on the degree and completeness of its processing, even oil.

Conclusion

The genetic code is universal. It means that genetic information is always borne by DNA whatever the organism (bacteria, fungus, plant or animal). Thus, DNA is easily recombined and transferred from one organism to another. Furthermore, DNA is a ubiquitous molecule as all the cells that form an organism contain the same DNA. In addition, DNA is a resistant molecule, in the sense that it is quite resistant to heat and acidity variations. All these properties represent advantages for the detection of this molecule in foodstu€s. Finally, very low amounts of DNA

may be ampli®ed thanks to PCR techniques, allowing easy identi®cation. Thus the PCR technique can target genes introduced into the genetically engineered plants thanks to a set of primers that amplify sequences from these cloned genes or from regulatory sequences linked to them. DNA fragments ampli®ed by PCR and having the expected size, are the signature of the sample which is being analysed. They indicate if foodstu€s are made from genetically-modi®ed or conventional plants. This enables us to determine, in case of positive result, with which genetically engineered species the food product is made.

References

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