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3.3.2 Dna-Based Testing Methods A GMO is usually defined as a living organism whose genetic composition has been altered by means of gene technology. This involves DNA isolation, defined DNA modification, and transfer of DNA into the genome of the target organism that successively becomes a GMO. This process is referred to as the transformation event. Normally, new gene functions are inserted into the GMO, but new techniques have been developed that make targeted knock-out of existing genes possible, among others in higher organisms, such as food plants. A typical insert (gene construct) in a GMO is composed of at least three elements: (1) the promoter element, which functions as an on/off switch for reading of the inserted/altered gene; (2) the gene that has been inserted/altered, which is coding for a specific selected feature; (3) the terminator element, which functions as a stop signal for reading of the inserted/altered gene. In addition, several other elements can be present in a gene construct and their function is usually to control and stabilise the function of the gene, demonstrate the presence of the construct in the GMO, or facilitate combination of the various elements of the construct. A gene construct must be integrated in the genome (the natural genetic background) of the organism to become stably inherited. The integration process itself is complex and largely beyond human control and in addition to one or more copies of the construct, fragments of the constructs may become inserted and stably integrated into the recipient genome, depending on the transformation strategy. Other breeding techniques than gene technology are used to modify DNA and increase genetic variability, such as chemical and irradiation mutagenesis. Although these techniques also involve genetic modifications, they do not involve the set of techniques defined as gene technology. Therefore, in most countries (not Canada and US), such techniques were defined to be outside the regulatory framework for authorisation and labelling for GMOs emerging in the 1990s. However, there are several scientific issues in common for GMOs and varieties established by other 1 breeding techniques. Common for all these technologies is that the genetic material is modified with the objective of creating new and more useful varieties. A major difference is the degree of targeting. Gene technology is considered a highly targeted technology, which may involve few to moderate numbers of changes and mainly tend to seek modification at a single locus, whereas alternative mutagenic technologies usually create a large number of more or less random changes. On the other hand, a unique feature of gene technology is that it allows for transfer of genetic material across species barriers. The degree of targetedness is of central relevance for the detectability, since better knowledge of the genetic changes means that it is easy to develop methods to detect the genetic modifications. DNA detection methods for GM foods rely on the complementarity of two strands of DNA double helix that hybridize in a sequence-specific manner. The DNA that has been engineered into a crop consists of several elements that govern its functioning. They are typically a promoter sequence, structural gene and a stop sequence for the gene. The availability of sufficient quantities of pure and intact DNA is always a crucial point in DNA-based methods, i.e. for polymerase chain reaction (PCR), DNA sequencing, Southern blotting, and microarrays. The same is true for the DNAbased methods for detection of genetically modified (GM) food. According to the European legislation, food products must be labeled as genetically modified, if GM compounds are present in proportion higher than 0.9%. GM food means food containing, consisting of or produced from genetically modified organisms (GMO). The majority of the existing methods for the detection of GM food is based on testing for the presence of recombinant DNA, or on the detection of novel expressed proteins. Nearinfrared (NIR) spectroscopy is also applicable, if there are enough differences between the conventional and the engineered food. DNA-based methods include Southern blotting, conventional qualitative PCR, quantitative competitive PCR, and qualitative or quantitative real-time PCR. During the production chain, food passes several physical, biological, and chemical processes, which mostly negatively influence the quantity and quality of available DNA. Three different approaches for DNA isolation from plant material and plant-derived products are the most widely used for GM food detection: the cetyltrimethylammonium bromide (CTAB) method, DNA-binding silica columns in form of various commercial kits and a combination of both. The existing methods for 2 DNA isolation from food cannot always fulfill the expectations regarding quantity and quality of isolated DNA. Furthermore, they usually require only up to 100 mg of food sample and are difficult to scale-up. Chromatographic methods were traditionally used for the isolation of proteins from various sources, such as microbial culture, animal tissue and plants. More recently, with emerging techniques in biotechnology and bioengineering, the isolation of recombinant proteins, peptides, carbohydrates and nucleic acids has gained importance. The supports for stationary phases applied in chromatography of large biomolecules must fulfill some distinct criteria, different from those for the chromatography of lowmolecular-mass biologically non-active molecules. The major problem is the restricted access of the biomolecules to the small pores of the classical porous chromatographic supports. The so-called wide-pore silica columns opened a broad field of new applications in the chromatography of biomolecules. Some problems such as the slow mass transport resulting in peak broadening and low recovery still remained unsolved. Another approach, which allowed overcoming the problem of mass transport, is the application of micropellicular supports characterized by a spherical fluid-impervious core of support material, covered by a thin retentive layer of stationary phase. Because of the lack of pores, the surface area is low, but rapidly and efficiently available for the mobile phase and the sample molecules. In the late 1980s, a novel type of supports has been introduced, called monoliths. Monoliths are constituted of a single piece of continuous and highly porous material, which is arranged in a way to form a network of highly interconnected channels. In such media, the mobile phase is forced to flow trough the pores. As a consequence, the mass transport is enhanced by convection, what contribute to minimize the void volume and the peak broadening. Five major chromatographic modes are used for the separation of nucleic acids: sizeexclusion, anion-exchange, mixed-mode, ion-pair reversed-phase and slalom chromatography. Among theme anion-exchange chromatography combined with micropellicular supports, is described as the most prominent technique so far. At neutral pH, the hydrophobic organic bases of the double-stranded DNA molecule are inside, whereas the two sugar-phosphate chains spiral down the outside of the double-helix structure. The sugar-phosphate chains create a poly-anionic, highly hydrated, and hydrophilic surface to the solvent allowing the chromatographic separation in anionexchange mode. CIM (Convective Interaction Media; BIA Separations, Ljubljana, 3 Slovenia) monolithic columns allow fast and flow-unaffected separation of several biomolecules, including nucleic acids. Anion-exchange chromatography with CIM disks allows the separation of oligonucleotides of different size and also the isolation of plasmid DNA from a complex matrix, i.e. E. coli cell lysate. Bacterial genomic DNA of size up to 200 kbp can be also separated on CIM monoliths. The specificity of currently available DNA based methods can be divided into four categories: 1) screening methods that can detect a wide range of GMO without identifying the GMO, 2) screening methods that can detect a certain type of genetic modifications, 3) construct specific methods that sometimes can be used to identify the GMO, 4) transformation event specific methods that can be used to identify the GMO with special exceptions mentioned below. In addition PCR-based GMO analyses usually include testing for presence of DNA from the particular species of interest, e.g. soybean DNA. Sometimes (in the absence of amplifiable DNA from the particular plant species) GMO analyses also include testing for presence of amplifiable (multicopy) DNA from plants or Eukaryotes, e.g. chloroplast DNA or nuclear ribosomal small subunit genes (18S like). The promoter and terminator elements used to transform most of the currently approved genetically modified plants are the Cauliflower Mosaic Virus promoter (P-35S) and the Agrobacterium tumefaciens nopaline synthase terminator (T-Nos). Although, other promoters and terminators have also been used, almost all GM plants contain at least one copy of the P-35S, T-35S and/or the T-Nos as a part of the gene construct integrated in its genome. Consequently, methods detecting one of these elements are popular for screening purposes (category 1). One problem with these methods is that the elements they detect are from naturally occurring virus and bacteria which are often present in fresh vegetables or the environment in which they are grown. Such elements therefore pose a significant risk of yielding false positive results. The various genes inserted in a GMO may characterize a group of GMO, although they may not identify the GMO. Detection of the synthetic specific gene coding for the 4 Bacillus thuringiensis endotoksin CryIA(b) demonstrates the presence of a genetically modified maize, but the gene has been used in more than one GMO. Consequently, category 2 methods like those that can detect the CryIA(b) gene can tell us more than the category 1 screening methods, but will not be suitable for identification of the specific GMO. The synthetic CryIA(b) gene has been integrated with different specific regulatory elements (promoters and terminators) in the various GMO containing the gene. Currently it is therefore possible to identify the GMO with category 3 detection methods targeting the junctions where the gene and regulatory elements are fused. However, in the future, even these junctions may be found in more than one GMO. With currently available technology it has not yet been possible to control where in the genome of a plant the insert is integrated. If the same insert is integrated into the genome of the same type of organism several times, the likelihood of integration of the insert in the same place in the genome two times is usually negligible. Consequently, the junction between the integration site and the insert will be unique for each transformation event. Category 4 methods detect these regions and will remain specific for the transformation even when the same construct has been integrated into the same plant species many times. However, category 4 methods are under development, and none has yet been published. When two GMO are crossed, e.g. two different approved genetically modified maize cultivars, the resulting hybrid offspring may possess the genetic modifications from both parent cultivars. This phenomenon is called "gene stacking". In the U.S.A. this type of hybrid GMO is not regulated, because both parent cultivars are approved. In the European Union, however, the hybrid is considered to be a new GMO and requires separate approval. None of the above four categories of analysis methods will be able to identify cases of gene stacking. Instead, cases of gene stacking will give results indistinguishable from the separate detection and identification of each of the parental cultivars in the sample. Table 1. Examples of published methods to detect GMO derivatives grouped according to categories of specificity Type of method Target sequence Reference 5 Method to detect plant derived DNA Chloroplast tRNALeu gene (trnL) intron Taberlet & al., 1991 Corn/maize single copy invertase gene Ehlers & al., 1997 Soybean single copy lectin gene Meyer & al., 1996 Tomato single copy polygalacturonase gene Busch & al., 1999 Cauliflower mosaic virus promoter (P-35S) Pietsch & al., 1997 Nopaline synthase terminator (T-Nos) Pietsch & al., 1997 bar (phosphinotricin acetyltransferase) gene Ehlers & al., 1997 CryIA(b) gene (synthetic) Ehlers & al., 1997 Methods to detect specific plant species Screening methods (category 1) Gene specific methods (category 2) Vaïtilingom & al., 1999 Construct specific methods (category 3) Bt11 maize: junction alcohol dehydrogenase 1S intron IVS6 (enhancer) - CryIA(b) gene Matsuoka & al., 2001 Bt176 maize: junction CDPK (calcium dependent protein-kinase) promoter synthetic CryIA(b) gene Hupfer & al., 1998 GA21 maize: OTP (enhancer) - epsps gene (RoundupReady tolerance) Matsuoka & al., 2001 Mon810 maize: junction P-35S - heat shock protein (hsp) 70 intron I (enhancer) Zimmermann & al., 1998 Mon810 maize: junction hsp 70 intron CryIA(b) gene Matsuoka & al., 2001 RoundupReady®: junction P-35S - Petunia hybrida CTP (chloroplast transit peptide) Wurz & Willmund, 1997 T25 maize: junction pat (phospinotricin acetyltransferase) gene - T-35S Matsuoka & al., 2001 Zeneca tomato: junction T-Nos - truncated tomato polygalacturonase gene Busch & al., 1999 6 Event specific methods (category 4) Bt11 maize: junction host plant genome integrated recombinant DNA Zimmermann & al., 2000 RoundupReady® soybean: junction host plant Berdal & Holstgenome - integrated recombinant DNA Jensen, in press Taverniers & al., in press Terry & Harris, in press 3.3.2.1 Southern blot The method involves fixing isolated sample DNA onto nitrocellulose or nylon membranes, probing with double-stranded (ds)-labeled nucleic acid probes specific to the GMO, and detecting hybridization radiographically, fluoremetrically or by chemiluminescence. Earlier probes were labeled with 32 P. However, nonradioactive fluorescein-labeled DNA, digoxigenin-, or biotin-labeled DNA probes, with sensitivity equal to 32 P probes, were recently used, obviating the need for radioactivity in the testing laboratory. These nonradioactive probes reduced detection to <1 h, as opposed to 24 h labeling required by 32 P. However, because only one probe is used, and no amplification is carried out, this method is considered less sensitive than PCR, which employs DNA of two primers. Recently, an alternative Southern blot technology has been attempted with near infrared (NIR) fluorescent dyes (emitting at ~700 and 800 nm) coupled to a carbodiimide-reactive group and attached directly to DNA in a 5 min reaction. The signals for both dyes are detected simultaneously (limit in the low zeptomolar range) by two detectors of an infrared imager, something not yet possible with conventional radioactive or chemiluminescent detection techniques. 3.3.2.2 Polymerase Chain Reaction 7 The investigation of an organism's genome was greatly enhanced during the early 1970s with the development of recombinant DNA technology. This technology allows for In vivo replication (amplification) of genomic DNA regions that are covalently linked with bacterial plasmid or virus clones. In 1985-86, a second major development occurred at Cetus Corporation, USA, where researchers developed an in vitro method for the amplification of DNA fragments, referred to as the polymerase chain reaction (PCR). The idea for PCR is credited to Kary Mullis, who along with five other researchers demonstrated that oligonucleotide primers could be used specifically to amplify specific segments of genomic DNA or cDNA. Mullis was awarded the Nobel Prize in chemistry in 1993 for his contribution to the development of PCR. This PCR is based on the features of semiconservative DNA replication carried out by DNA polymerases in prokaryotic and eukaryotic cells. PCR results in the selective amplification of a chosen region of a DNA molecule. In this technique, DNA molecule is not cloned in a bacterial plasmid or virus. The only requirement is that the sequence at the borders of the selected DNA region must be known so that two short oligonucleotides can anneal to the target DNA molecule for amplification. These oligonucleotides delimit the region that will be amplified. In normal DNA replication within a cell, DNA duplex opens up and a small single-stranded RNA primer is synthesized to which DNA polymerase adds further nucleotides. In PCR, a specific region of DNA, which is flanked by two oligonucleotide (deoxy) primers that share identity to the opposite DNA strands, is enzymatically amplified. Amplification of selected region from a complex DNA mixture is carried out in vitro by the DNA polymerase I from Thermus aquaticus, a bacterium that lives hot springs, This amplification is achieved by a repetitive series of cycles involving three steps: 1. Denaturation of a template DNA duplex by heating at 94°C. 2. Annealing of oligonucleotide primers to the target sequences of separated DNA strands at 55-65°C. 3. DNA synthesis from the 3'-OH end of each primer by DNA polymerase at 72°C. 8 By using repetitive cycles, where the primer extension products of the previous cycle serve as new templates for the following eye the number of target DNA copies has the potential to double each cycle (Fig.2). This technique became important with the discovery of Taq DNA polymerase by Kary Mullis. Taq DNA polymerase is a thermostable enzyme, which retains activity even after denaturation by heat treatment at 94°C, whereas the DNA polymerase initially used for amplification was the Klenow fragment of E. coli DNA polymerase I that could not withstand the denaturation step. Earlier, amplification was achieved by transferring samples in water baths maintained at different temperatures and adding fresh enzyme during each cycle of amplification. Presently automated thermal cycling apparatus are available that can amplify a millionfold target DNA sequence with just 20 PCR cycles in few hours. In theory, each amplification cycle should double the number of target molecules, resulting in an exponential increase in the PCR product, However, even before substrate or enzyme becomes limiting, the efficiency of exponential amplification is less than 100% due to suboptimal DNA polymerase activity, poor primer annealing, and incomplete denaturation of the templates. PCR efficiency can be expressed as; PCR product yieldn= (input target amount) x (1 + % efficiency) cycle number This equation can be used to calculate that ~26 cycles are required to produce 1 µg of PCR product from 1 pg of a target sequence (10 6 amplification) using an amplification efficiency value of 70%. [1 µg PCR product = (1 pg target) x (1 + 0.7)26.] Polymerase chain reaction is useful because of automated instrumentation. A standard PCR machine is basically a thermal cycling instrument. The minimum requirement for a thermal cycler is that it be capable of rapidly changing reaction tube temperature to provide the optimal conditions for each step of the cycle. There are three basic designs, but a design represented by the MJ Research DNA engines™, is the most common. It utilizes the heating/cooling pump principle of the Pelletier effect, which is based on 9 heat exchange occurring between two dissimilar surfaces that are connected in series by electric current, Figure.3 shows the temperature profile for a typical PCR reaction cycle run on a thermal cycler. The ramp time and temperature accuracy are the most important parameters that must be controlled by the thermal cycler. A hot start is a method that can be used to increase primer specificity during the first round of DNA synthesis. In this procedure, the Taq polymerase is held inactive at temperatures below the annealing optimum (<55°C) to avoid extending mismatched primers. The figure also shows a graph depicting the amount of PCR product produced as a function of cycle number. The PCR efficiency equation applies only to the exponential phase, which represents the linear range of the reaction when plotted on a log scale. The plateau effect results from enzyme limitations due to decreased enzyme activity from repeated exposure to 95°C and from stoichiometric limitations of active enzyme molecules, relative to DNA templates, within the time frame of extension period. 3.3.2.2.1 Procedure The polymerase chain reaction is used to amplify a segment of DNA that lies between the two regions of known sequence where two oligonucleotides (deoxy) as primers can bind the opposite strands of DNA due to the complementary nature of base sequences. Taq DNA polymerase catalyzes the amplification reaction (Fig.2). The template DNA is first denatured by heating at 94°C. The reaction mixture is then cooled to a temperature that allows the primers to anneal to their target sequences. These annealed primers are then extended (i.e. synthesis of DNA) with Taq DNA polymerase. The cycle of denaturation, annealing, and DNA synthesis is repeated many times, because the product of one round of amplification serves as template for the next, each successive cycle essentially doubles the amount of the desired DNA product. The major product of this exponential reaction is a segment of double-stranded DNA whose ends are defined by the 5' termini of the primers and whose length is defined by the distance between the primers. The products of first round of amplification are heteroge- neously sized DNA molecules, whose lengths may exceed the distance between the binding sites of the two primers. In cycle 2, the original strands and the new strands from cycle I are separated, yielding a total of four primer sites vith which primers anneal. The primers that are hybridized to the new strands from cycle I are extended by polymerases as far as the ends of the template, leading to a precise 10 copy of the target region. In cycle 3, double-stranded DNA molecules are produced that are precisely identical to the target region. The original DNA strands and the variable extended DNA strands will continuously be produced from o exponential doubling of the original template but at a linear rate and become negligible after the exponential increase of target fragments (Table.2). The reaction can be continued for 25-45 cycles. Under normal conditions, the amount of Taq DNA polymerase becomes limiting after 30 cycles of amplification. If further amplification is required, then a sample of the amplified DNA can be diluted and used as a template for further rounds of synthesis in a fresh PCR reaction. 11 Figure 2. Schematic Diagram Of The Polimerase Chain Reaction 12 Figure 3. a. Temperature Profile of a Typical PCR Cycle, b. Accumulation of PCR target molecules as a function of cycle number. 13 Table 2. PCR amplification of a target fragment with increasing number of the cycles 3.3.2.2.2 Components in a polymerase chain reaction 1. Oligomer primers: The amplification product of a PCR reaction is determined by the sequence of PCR primers. The primers anneal to complementary sequences on the DNA template and thereby determine the boundaries of the amplified product. Usually primers are used at a concentration of 1 µM, which is sufficient for at least 30 cycles of amplifications. It is assumed that the Taq DNA polymerase begins to work as soon as priming oligomers be- come bound to their templates at low temperatures (35-55°C). Once a DNA target has been chosen, there are several rules of thumb for primer design that are important to consider, 14 (a) Primer length: Primers for the PCR should be 16-24 nucleotides long, and are generally, referred to as primers 1 and 2 for the two ends of target sequence. Primers of this much length maintain specificity (chance of finding the same sequence by chance is only 1 in 420) and provide sufficient base pairing for stable duplex formation, (b)Duplex stability: Both the primers in a PCR reaction should have similar melting temperatures (Tm) to ensure that they will have the same hybridization kinetics during the temperature annealing phase. Primers with an overall G + C content of 4555% are most common. The Tm of each oligonucleotide can be calculated using an empirical formula that takes into account base composition and the effect of nearest neighbor interactions (the Tm of AAG is different from AGA). For primers with a low GC content, it is desirable to choose a long primer so as to avoid a low melting temperature. (c)Noncomplementary primer pairs: The two primers should not share complementarity at the 3' ends because they will give rise to primer dimer products. For example, if one primer has the sequence 5' — …GGCG — 3' and the other primer has a terminal sequence 5' — ...CCGC — 3', then they can form a short hybrid that will become a substrate for DNA synthesis. Once the primer dimer product is formed, it is a competing target for amplification, (d) No hairpin loops: Each primer must be devoid of palindromic sequences that can give rise to stable intrastrand structures that limit primer annealing to the template DNA. (e) Optimal distance between primers: This rule is specific for application, but for most diagnostic PCR assays, it is best when the opposing primers are spaced 150500 bp apart, (f) Sequences with long runs (i.e. more than three or four) of a single nucleotide should be avoided. Presently, a variety of computer algorithms has been developed, which takes all these parameters into account, and the researcher only needs to provide a DNA target sequence. 15 2. Amplification buffer: The standard buffer for PCR reaction contains KCI, TrisCI, and 1.5 mM magnesium chloride. When incubated at 72°C, the pH of the reaction drops by more than a unit. To maintain the pH, presence of divalent magnesium cations is critical. 3. Deoxyribonucleoside triphosphates: dNTPs are used at a saturating concentration of 200 µM for each dNTR 4. Target sequence: Template DNA containing the target sequences can be added in a single or double- stranded form. Linear target sequences are amplified better as compared to closed circular DNAs. Purity of template is flexible and even crude DNA extracts (e.g. pulp of a fossil tooth, a hair follicle) can be used as long as the addition of template does not inhibit the activity of polymerase. The concentration of the target sequence in the PCR reaction is generally in nanograms (5-100 ng). 5. Taq DNA polymerase: Two forms of Taq DNA polymerase are available: the native enzyme purified from Thermus aquaticus and a genetically engineered form synthesized in E. coli (Ampli Taq-™). Taq DNA polymerase is the most frequently used and preferred en- zyme. The original PCR protocol used the Klenow fragment of E. coli DNA polymerase I to perform the primer extension reaction. However, this meant that fresh enzyme had to be added after each round of denaturation because this enzyme is easily heat inactivated. Another problem with E. coli DNA polymerase is that the optimal activity level of the enzyme is 37°C, which greatly limits the specificity of the reaction due to degenerate primer annealing at this low temperature. Both these problems were solved by the use of Taq DNA polymerase. This enzyme retains activity even after repeated exposure to a temperature of 95°C, and is also fully active at ~75°C, which essentially eliminates degenerate heteroduplex formation. More recently, several other thermostable DNA polymerases have been isolated and characterized that offer advantages for specialized PCR assays (Table.3). The biggest difference between these enzymes is their inherent 3'->5' exonuclease proof reading activity, which is important if the sequence of PCR product must be error free. DNA polymerases with 3'->5' exonuclease 16 activity are Tli from Thermococcus litoralis and Pfu from Pyrococcus furiosus, which have a 10-100 times lower error rate than DNA polymerases lacking this activity. Taq DNA polymerase and E. coli DNA polymerase I encode a 5'-»3' exonuclease activity.Two other parameters are processivity (number of nucleotides polymerized before template dissociation) and extension rate (nucleotides polymerized/second). Some thermostable DNA polymerases can use RNA templates as a substrate, which can be useful for PCR applications that require a separate cDNA synthesis reaction using viral reverse transcriptase. An enzyme of this is the recombinant form of Tth polymerase (rTth) from Thermus thermophilus, which can catalyze high temperature transcription of RNA in the presence of MnCI2. Approximately 2 units of either of the enzymes are required to catalyze a typical PCR. Addition of excess enzyme may lead to amplification of nontarget sequences. Table 3. Characteristic features of a different thermostable DNA polymerases Both forms of the polymerase (Taq and Ampli Taq™) carry a 5'->3' polymerase dependent exonuclease activity, but they lack a 3'->5' exonuclease activity. It means that Taq poly- merase does not correct the incorporation of mismatched bases. The reaction is carried out in a sterile 0.5-ml microfuge tube by mixing the components in the order of sterile water, amplification buffer, dNTPs, primers 1 and 2, template DNA and finally the Taq DNA polymerase. Total volume can be 50 or 100 µl The reaction mixture is overlayered with 2-3 drops of mineral oil. This prevents evaporation of the sample during repeated cycles of heating and cooling. But now PCR 17 thermal cyclers are available with heated lid; thus, mineral oil is not required in those reactions. Also, reactions can be carried out in a much smaller volume of 10-25 µl. The amplified DNA product is detected by agarose gel electrophoresis, followed by staining with ethidium bromide. 18