DNA-based analysis targets the novel DNA sequences introduced into the crop. These methods show the absence or presence of novel plant material in a sample and some of them can also measure the relative quantity (percentage) in a tested sample.
2.1 DNA amplification-based methods (PCR)
Amplification techniques involve denaturation of the double stranded nucleic acid followed by annealing of a short oligonucleotide (primer) and primer extension by a DNA polymerase. The most common technique is the polymerase chain reaction (PCR) technique, employing a thermo-stable DNA polymerase.
PCR is the most commonly used technique for GMO detection. Figure 19 details the different levels of specificity of GMO detection possible with PCR technology (from screening to construct-specific and event-specific), depending on the type of DNA sequence information available.
Nucleotide sequence specific oligonucleotides, binding to the target DNA to the left and to the right of the target site, allow an enzyme to prolong the oligonucleotide primers and thereby to amplify specifically the DNA fragment between the primers. Repeated cycles of the reaction lead to a logarithmic amplification of the fragment. The design of specific primers depends on knowledge of the precise and comprehensive DNA sequence information of the actually integrated DNA.
If the method is to specifically detect and identify a certain transformation event (event-specific method), information about the inserted DNA sequence and about the 3’ and 5’ flanking plant genome sequences is required (Fig. 2).
For element-specific, PCR-based screening, and construct-specific detection, the DNA sequences of the inserted elements and gene constructs are targeted, respectively.
PCR-based detection and particularly the quantitative measurement of the GM content in a sample actually involves the use of two PCR systems, one for determination of the inserted GM-derived DNA sequence and another system specific for an endogenous, plant-taxon specific reference gene sequence (Fig. 20). The latter also serves as a control for the quality and quantity of the extracted DNA.
2.1.1 Conventional qualitative PCR
Conventional PCR methods are mainly used for qualitative testing to obtain yes/no answers concerning the presence of GM plant material. PCR products are analysed by agarose or polyacrylamide
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gel electrophoresis and visualised using UV fluorescence with ethidium bromide as fluorophore or by other means.
It may be necessary to confirm GM-positive test results by further analyses, either by restriction analyses, Southern hybridisation or DNA sequencing.
The important performance criteria for qualitative PCR methods are the sensitivity in detecting the DNA sequences and the specificity for the targeted DNA segment. At optimal reaction conditions a limit of detection (LOD) of 1 - 10 copies of the target sequence can be achieved in less than 40 PCR cycles. Practically the LOD of the PCR method should allow that the presence of the target sequence is detected in at least 95 % of the time, with less than 5 % false negative results. The length of the amplified product influences the PCR performance and should therefore be selected in a way that it matches to the size range of DNA fragments which can be extracted from the sample matrix. For raw materials like seeds or leaves containing less fragmented DNA a broader range of PCR product size up to maximally 250 bp is applicable, whereas for processed food or feed with higher DNA fragmentation the PCR product should be ideally 80 - 150 bp. The specificity of the method should be tested theoretically by sequence similarity search with the primer sequences against nucleic acid sequence databases and empirically by testing the target event(s), very similar non-target events and different non-modified plants in order to confirm that the primers can discriminate between the target and closely related non-target sequences. For reference gene-specific PCR methods, different varieties should be tested to demonstrate that the target sequence is conserved between different plant lines.
2.1.2 Quantitative Real-Time PCR
The most preferred technique to quantify GM material in a sample is Real-Time PCR. It allows the detection and measurement of increasing fluorescence proportional to the amount of amplification products generated during the PCR process. Of the various chemistries TaqMan fluorogenic probes are most commonly applied in Real-Time PCR-based detection and quantification of GM plant materials.
Real-Time PCR is mainly used for quantification purposes, but it is increasingly utilised also for qualitative testing to screen or to identify the GM event.
The limit of quantification (LOQ) of a Real-Time PCR method depends on the optimisation of the PCR detection method and on the accepted standard deviation of the measurement. The LOQ is experimentally determined during method validation and should reach 30 - 50 target molecules, which is close to the theoretical prediction. The LOD / LOQ values depend primarily on the characteristic plant genome size (C value).
Note : the EU-RL GMFF and the ENGL have developed various guidance documents on PCR methods, including in particular the document on “Definition of Minimum Performance Requirements for Analytical Methods of GMO Testing” (available at http://gmo-crl.jrc.ec.europa.eu/default.htm) which defines the acceptance criteria to be met before a method can enter the EU validation process. Parameters addressed in this guidance document include Applicability, Practicability, Specificity, Dynamic Range, Trueness, Amplification Efficiency, Precision, LOD, LOQ and Robustness.
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Figure 19 details the different levels of specificity of GMO detection possible with PCR technology (from screening to construct-specific and event-specific), depending on the type of DNA sequence information available.
2.1.3 Conclusions for detection by PCR-based methods
Any PCR-based method relies on the availability of a certain minimum of information about the target DNA sequence. Some information needs to be known about the inserted DNA sequence and about the 5’ and/or 3’ neighbouring genomic DNA sequence in order to allow the identification of an intentional genetic modification (see further details below).
Without prior knowledge, reliable identification of a genetic modification is not possible even with the most sophisticated available methods for DNA analysis.
PCR-based analytical methods for the detection of intentionally modified DNA sequences provide high sensitivity and specificity. PCR supports the development of specific methods that allow the detection as well as the identification of intentionally modified DNA, i.e. plants with known intentional modifications can be differentiated for instance from plants presenting similar phenotypes and from plants possibly presenting a similar DNA modification through natural mutation.
2.1.3.1 Insertions larger than 80 bp
For the detection and the identification of an insert, the primers and probe need to be designed within the insert. Large inserts can be detected and identified when at least 80 bp of the inserted sequence is known.
Figure 19: Schema of a transformation construct comprising seven elements inserted into a plant genome through a certain transformation event and, therefore, flanked by specific DNA sequences of the plant genome.
Arrows of the upper four rows indicate regions suitable for element-specific detection. Such screening assays target widely used genetic elements like promoters.
Arrows in the following three rows in the middle indicate regions suitable for construct-specific detection. Construct-specific assays are designed to comprise a junction between different elements of the inserted sequence.
Arrows in the two rows at the bottom indicate regions suitable for event-specific detection. Event-specific assays are the most specific ones and are constructed over a junction between the host and the inserted sequences with specific primers for the inserted gene and the flanking genomic sequence.
An example for a reference gene is indicated. The two triangles at the right hand side indicate a gradient of suitability for screening, identification, and quantification. .
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For event-specific identification, a sufficient part of the sequence of the insert as well as a part of the adjacent sequence must also be known, in order to be able to design an event-specific primer pair and a probe. This information is a prerequisite for an unambiguous identification of an intentional genetic modification.
2.1.3.2 Short insertions
PCR-based methods are also capable to detect and identify short insertions of less than 80 bp. In this case specific primers are designed in order to bind to sequences including the insert and its flanking regions sites or to bind only to sequences directly flanking the insert. Irrespective of the number of modified base pairs, the specific primers should be at least approximately 20 nucleotides long and specific in sequence for the modification and its direct vicinity. In order to identify a short intentional modification and to differentiate it from a possible natural mutation, information on the modified sequence and the nucleotide sequence in its direct vicinity is required for the design of specific primers.
2.1.3.3. Modification of one or a few nucleotides
Intentional modifications of a single or a few nucleotides can in principle be detected. Information on the site of the modification and the nucleotide sequence in its direct vicinity of approximately 20 bp (including the site of modification) is necessary to ensure in principle the uniqueness of the sequence forming the newly created junction in the genome. For the amplification of this unique sequence by PCR further information upstream and downstream is required for the design of primers. If this 20 bp string matches with a repetitive sequence in the genome it cannot however unambiguously characterise the location of the modification.
2.1.3.4 Deletions
Deliberate modifications by deletions can also be detected in a similar way as described for modifications by short insertions. Information on the site of the deletion and the nucleotide sequence in its direct vicinity of approximately 20 bp including the site of deletion is necessary to ensure in principle the uniqueness of the sequence forming the newly created junction in the genome. For the amplification of this unique sequence the same requirement applies as for modification of a single or a few nucleotides.
If this 20 bp string matches with a repetitive sequence in the genome it cannot however unambiguously characterise the location of the modification.
2.2 DNA Sequencing
DNA sequencing allows determining the order of the nucleotide bases adenine, guanine, cytosine, and thymine in a DNA strand.
DNA sequencing is most commonly done on PCR amplified or cloned DNA fragments.
Determining the DNA sequence is useful in basic research studying fundamental biological processes, as well as in applied fields such as diagnostic and detection or forensic research.