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Development of a peptide nucleic acid polymerase chain reaction clamping assay for semiquantitative evaluation of genetically modified organism content in food
Abstract
In the present study a peptide nucleic acid (PNA)-mediated polymerase chain reaction (PCR) clamping method was developed and applied to the detection of genetically modified organisms (GMO), to test PCR products for band identity and to obtain a semiquantitative evaluation of GMO content. The minimal concentration of PNA necessary to block the PCR was determined by comparing PCRs containing a constant amount of DNA in the presence of increasing concentration of target-specific PNA. The lowest PNA concentration at which specific inhibition took place, by the inhibition of primer extension and/or steric hindrance, was the most efficient condition. Optimization of PCR clamping by PNA was observed by testing five different PNAs with a minimum of 13 bp to a maximum of 15 bp, designed on the target sequence of Roundup Ready soybean. The results obtained on the DNA extracted from Roundup Ready soybean standard flour were verified also on DNA extracted from standard flours of maize GA21, Bt176, Bt11, and MON810. A correlation between the PNA concentration necessary for inducing PCR clamping and the percentage of the GMO target sequence in the sample was found.
... Table 1 Examples of PCR based detection methods. a Species Event OECD unique ID Tradename(s) Construct C, gene G or event E specific b Reference Cotton 281-24-236 DAS-24236-5 Widestrike E Baeumler et al. (2006), European Commission (2009) 3006-210-23 DAS-21023-5 Widestrike E Baeumler et al. (2006), European Commission (2009) GBH614 BCS-GH002-5 E European Commission (2009) GK12 C Cheng et al. (2007) GK19 C Yang et al. (2005a) LLCOTTON25 ACS-GH001-3 E European Commission (2009) ME (QL) Kim et al. (2008b) Mon531 MON-00531-6 C Yang et al. (2005a) E European Commission (2009), Yang et al. (2005b) Mon1445 MON-01445-2 E European Commission s(2009), Yang et al. (2005b) ME (QL) Kim et al. (2008b) Mon15985 MON-15985-7 BollGardII C Lee et al. (2007) E European Commission (2009) ME (QL) Kim et al. (2008b) Mon88913 MON-88913-8 C Lee et al. (2007) ME (QL) Kim et al. (2008b) SGK321 C Yang et al. (2005a) Maize (corn) 3272 SYN-E3272-5 E European Commission (2009) 59122 DAS-59122-7 E European Commission (2009) Bt10 – E (QL) European Commission (2009), Watanabe et al. (2007) Bt11 SYN-BT011-1 C Brodmann et al. (2002), Matsuoka et al. (2002), Peano et al. (2005b) ...
... MC Rudi et al. (2003) E (comp) Zimmermann et al. (2000) E European Commission (2009), Rønning et al. (2003), Taverniers et al. (2005) ME (QL) Hernandez et al. (2005a,b), Xu et al. (2007) Bt176 SYN-EV176-9 G Bordoni et al. (2004), Ehlers et al. (1997) G (comp) Garcia-Canas et al. (2004a,b) C Brodmann et al. (2002), Matsuoka et al. (2002), Peano et al. (2005b),Vaïtilingom et al. (1999) ...
... s E (QL) Windels et al. (2003) DBT418 MG Rudi et al. (2003) DLL25 C Matsuoka et al. (2002) Event 32 DAS-59132-8 E European Commission (2009) GA21 MON-00021-9 MG Rudi et al. (2003) C Hernandez et al. (2004), Matsuoka et al. (2002), Peano et al. (2005b) ...
This paper presents an overview of GMO testing methodologies and how these have evolved and may evolve in the next decade. Challenges and limitations for the application of the test methods as well as to the interpretation of results produced with the methods are highlighted and discussed, bearing in mind the various interests and competences of the involved stakeholders. To better understand the suitability and limitations of detection methodologies the evolution of transformation processes for creation of GMOs is briefly reviewed.
... They are widely used to amplify target DNA fragments corresponding to marker genes, promoter and terminator sequences , or to transgene-host integration junctions (Ahmed, 2002; Anklam, Gadani, Heinze, Pijnenburg, & van den Eede, 2002; Corbisier et al., 2007; Miraglia et al., 2004; Rodriguez-Lazaro et al., 2007). Beyond end-point and real-time PCR, several innovative, fast and reproducible methods have been recently developed for the identification of different GMO events (Bordoni, Germini, Mezzelani, Marchelli, & De Bellis, 2005; Germini et al., 2005; Kim, Chae, Chang, & Kang, 2005; Peano et al., 2005; Rossi et al. 2007; Xu et al., 2006). Among these, DNA biosensors based on several transduction principles (Gambari & Feriotto, 2006; Giakoumaki et al., 2003; Kalogianni, Koraki, Christopoulos, & Ioannou, 2006; Leimanis et al., 2006; Mannelli, Minunni, Tombelli, & Mascini, 2003; Minunni et al., 2005; Passamano & Pighini, 2006) are methods able to detect target sequences in complex media and in real time. ...
... c o m / l o c a t e / f o o d c h e m gene, naturally present in the genome of the analyzed species (i.e., zein in Zea mays and lectin in Glycine max) (Meyer, Chardonnens, Hubner, & Luthy, 1996; Meyer & Jaccaud, 1997 ). Despite the interesting aspects of these studies, most of the reported work refers to raw material, such as flours or seeds, or to the final product of food processing (Aarts, van Rie, & Kok, 2002; Ahmed, 2002; Di Pinto et al., 2007; Engel, Moreano, Ehlert, & Busch, 2006; Germini et al., 2005; Passamano & Pighini, 2006; Peano et al., 2005). A systematic study on the traceability of GM DNA along a food processing chain has never been reported, and only laboratory model studies have been conducted on different processing procedures (Bauer, Weller, Hames, & Hertel, 2003). ...
... The DNA degradation can be caused, for example, by heating treatments (Ahmed, 2002) and exposure to pH changes (Aarts et al., 2002). Finally, another important factor which could affect the amplification step, leading also to false negative results should be taken into account, which is the presence of different inhibitors of the enzyme Taq polymerase such as proteins, fats, polysaccharides, polyphenolics and other compounds that may be present in DNA extracted from food matrices (Corbisier et al., 2007; Di Pinto et al., 2007; Lüthy, 1999; Peano et al., 2005). In this work we report for the first time the study of transgene traceability along a complete industrial soybean processing chain. ...
The development of analytical methods for genetically modified organisms (GMO) screening is of great interest. In particular, since even highly processed GMO-derived food products are covered by new European legislations, a great effort has been devoted to the application of the analytical tests to these products.This work describes a polymerase chain reaction-based qualitative screening assay and a biosensor-based approach to detect transgenes in a Roundup Ready® soybean processing line. Roundup Ready® soybean was specifically analyzed in eight types of processed materials – seeds, crushed seeds, expander, crude flour, proteic flour, crude oil, degummed oil and lecithin – all derived from the same initial source and produced during the manufacturing process. Specific combinations of primers were used to differentiate sequences from the whole insert. The amplification of “marker” fragments with a maximum length of 500 bp was successfully achieved both in raw material (seeds) and in partially (crushed seeds, crude and proteic flours) and highly (crude and degummed oils and fluid lecithin) processed materials.Moreover, the extraction procedure was optimised and the polymerase chain reaction-electrophoresis analysis has been implemented by a biosensor-based approach.
... Table 1 Examples of PCR based detection methods. a Species Event OECD unique ID Tradename(s) Construct C, gene G or event E specific b Reference Cotton 281-24-236 DAS-24236-5 Widestrike E Baeumler et al. (2006), European Commission (2009) 3006-210-23 DAS-21023-5 Widestrike E Baeumler et al. (2006), European Commission (2009) GBH614 BCS-GH002-5 E European Commission (2009) GK12 C Cheng et al. (2007) GK19 C Yang et al. (2005a) LLCOTTON25 ACS-GH001-3 E European Commission (2009) ME (QL) Kim et al. (2008b) Mon531 MON-00531-6 C Yang et al. (2005a) E European Commission (2009), Yang et al. (2005b) Mon1445 MON-01445-2 E European Commission s(2009), Yang et al. (2005b) ME (QL) Kim et al. (2008b) Mon15985 MON-15985-7 BollGardII C Lee et al. (2007) E European Commission (2009) ME (QL) Kim et al. (2008b) Mon88913 MON-88913-8 C Lee et al. (2007) ME (QL) Kim et al. (2008b) SGK321 C Yang et al. (2005a) Maize (corn) 3272 SYN-E3272-5 E European Commission (2009) 59122 DAS-59122-7 E European Commission (2009) Bt10 – E (QL) European Commission (2009), Watanabe et al. (2007) Bt11 SYN-BT011-1 C Brodmann et al. (2002), Matsuoka et al. (2002), Peano et al. (2005b) ...
... MC Rudi et al. (2003) E (comp) Zimmermann et al. (2000) E European Commission (2009), Rønning et al. (2003), Taverniers et al. (2005) ME (QL) Hernandez et al. (2005a,b), Xu et al. (2007) Bt176 SYN-EV176-9 G Bordoni et al. (2004), Ehlers et al. (1997) G (comp) Garcia-Canas et al. (2004a,b) C Brodmann et al. (2002), Matsuoka et al. (2002), Peano et al. (2005b),Vaïtilingom et al. (1999) ...
... s E (QL) Windels et al. (2003) DBT418 MG Rudi et al. (2003) DLL25 C Matsuoka et al. (2002) Event 32 DAS-59132-8 E European Commission (2009) GA21 MON-00021-9 MG Rudi et al. (2003) C Hernandez et al. (2004), Matsuoka et al. (2002), Peano et al. (2005b) ...
This paper presents an overview of GMO testing methodologies and how these have evolved and may evolve in the next decade. Challenges and limitations for the application of the test methods as well as to the interpretation of results produced with the methods are highlighted and discussed, bearing in mind the various interests and competences of the involved stakeholders. To better understand the suitability and limitations of detection methodologies the evolution of transformation processes for creation of GMOs is briefly reviewed.
... Physics [Shakal & Bernreuter, 1980] [Courteau et al, 2007] [McIntosh et al, 2004] [Garland et al, 2001] [Zhang et al, 2007] [ Kwon and Kubicki, 2004] [Yoshida et al, 2008] [Zhang et al, 1989] [Standish, 1993] [Daragan and Mayo, 1995] Biology [Gianola et al, 2006] [Mollet et al, 1997] [Noth and Benecke, 2005] [Peano et al, 2005] [Eisenrauch and Bamberg, 1990] [Kloczkowski et al, 2002] [ Kompalic-Cristo, 2004] [Battles et al, 1995] [Ounis et al, 2001] [Won and Min, 2010] [Moskovets et al, 2003] Anthropology [Wassenberg et al, 2003] [Keyton and Rhodes, 1997] [Aguilar, 2008] [Wicklund, 1995] [Crean and Wisher, 2000] [Cudney and VanTuyle, 2001] [Posner, 1998] [Jaeger, 1992] [Romero, 1994] [Babrow et al, 1994] Geosciences [Klose, 2006] [Rapalini, 2007] [Degtyarev et al, 2008] Chemistry [Lenglet et al, 1995] [Pizarro et al,2000] [Kounaves, 2003] [Arora et al, 1999] [Iannelli et al, 2006] [Leen and Mills, 1999] Psychology [Charlton, 2000] [Dillenbourg, 1996] [Kuckertz et al, 2012] [Bressler et al, 2006] [Meichi, 2003] [Charash and McKay, 2009] [Allegro, 1990] [Stevenson and Evans, 1994] [Rodden et al, 2010][Soni, 2011 Economy [Ozdincer and Ozyildirim, 2011] [Smith, 2003] [Starczak and Jakubiec, 2003] [Smith and Natesan, 1999] [Holcombe, 1992] [Durand, 2007] [Lonkani et al, 2012] [Stanley, 2000] [Gischer and Juttner, 2001] [Methanuntakul, 2010] Physical measure The distance dependence of ground motion relationships derived from close-in data is very sensitive to the ambiguous interpretation of distance when the station is a source dimension or less from the fault. The choice of the method for measuring distance can have a dramatic impact on the resulting ground motion curves [Shakal et Bernreuter, 1980]. ...
... The different probe and gene IDs corresponding to the two annotation releases generates uncertainties [Noth and Benecke, 2005]. PCR methods can sometimes be controversial and a post-PCR control has been shown to be often essential to confirm a sequence identity in case of ambiguous recognition of specific targets [Peano et al, 2005]. In some biological approaches ionophores were used for the demonstration of the electrogenic properties of the enzyme, which could lead to a problem of interpretation of electrogenicity [Eisenrauch and Bamberg, 1990]. ...
Text data is often seen as "take-away" materials with little noise and easy
to process information. Main questions are how to get data and transform them
into a good document format. But data can be sensitive to noise oftenly called
ambiguities. Ambiguities are aware from a long time, mainly because polysemy is
obvious in language and context is required to remove uncertainty. I claim in
this paper that syntactic context is not suffisant to improve interpretation.
In this paper I try to explain that firstly noise can come from natural data
themselves, even involving high technology, secondly texts, seen as verified
but meaningless, can spoil content of a corpus; it may lead to contradictions
and background noise.
... [30]], environmental microbiology [e.g. [31]], parasitology [32], and to detect genetically modified organism content in food [33], but not to our knowledge in diet studies. However, synthesis time for clamping probes is several weeks and these probes are also quite expensive. ...
... It is not known why the elongation arrest primer was unsuccessful. Von Wintzingerode et al. [31] and also Peano et al. [33] using PCR clamping and PNA probes found that elongation arrest clamping worked, but that competitive clamping (annealing inhibition) was more efficient, and the former hypothesised that this was due to interaction between the Taq polymerase and PNA and DNA. However, since elongation arrest primers allows the unmodified version of the universal primer to bind to the dominating sequence, and just inhibit the generating of its amplicons, perhaps the explanation of no PCR product is that the universal primers never found the rarer sequences. ...
Identification of DNA sequence diversity is a powerful means for assessing the species present in environmental samples. The most common molecular strategies for estimating taxonomic composition depend upon PCR with universal primers that amplify an orthologous DNA region from a range of species. The diversity of sequences within a sample that can be detected by universal primers is often compromised by high concentrations of some DNA templates. If the DNA within the sample contains a small number of sequences in relatively high concentrations, then less concentrated sequences are often not amplified because the PCR favours the dominant DNA types. This is a particular problem in molecular diet studies, where predator DNA is often present in great excess of food-derived DNA.
We have developed a strategy where a universal PCR simultaneously amplifies DNA from food items present in DNA purified from stomach samples, while the predator's own DNA is blocked from amplification by the addition of a modified predator-specific blocking primer. Three different types of modified primers were tested out; one annealing inhibiting primer overlapping with the 3' end of one of the universal primers, another annealing inhibiting primer also having an internal modification of five dI molecules making it a dual priming oligo, and a third elongation arrest primer located between the two universal primers. All blocking primers were modified with a C3 spacer. In artificial PCR mixtures, annealing inhibiting primers proved to be the most efficient ones and this method reduced predator amplicons to undetectable levels even when predator template was present in 1000 fold excess of the prey template. The prey template then showed strong PCR amplification where none was detectable without the addition of blocking primer. Our method was applied to identifying the winter food of one of the most abundant animals in the world, the Antarctic krill, Euphausia superba. Dietary item DNA was PCR amplified from a range of species in krill stomachs for which we had no prior sequence knowledge.
We present a simple, robust and cheap method that is easily adaptable to many situations where a rare DNA template is to be PCR amplified in the presence of a higher concentration template with identical PCR primer binding sites.
... This was suggested because of the complex interactions among the primer, DNA and Taq polymerase (von Wintzingerode et al. 2000). However, elongation arrest primers worked in some cases (Peano et al. 2005;von Wintzingerode et al. 2000;Yu et al. 1997). It was found that PCR parameters could be optimized to make elongation arrest work, such as designing a blocker with a higher Tm than the primer Tm (O'Rorke et al. 2012). ...
Planktonic protists, including both autotroph and heterotroph, have been recognized as a major contributor to primary production and consumers of bacteria, archaea and picophytoplankton. However, the understanding of protistan diversity is typically impeded by the large amount of metazoans when employing universal primers to environmental samples. In this study, we developed 3 blocking primers to inhibit the amplification of metazoan DNA in PCR. First, we optimized the design and concentration of blocking primers by using 2 metazoans and 3 protists in quantitative PCR. No significant difference (q > 0.05) was found in protistan community structure at phylum, family or OTU levels and genetic diversity between samples amplified with and without adding blocking primers, indicating that the blocking primer does not alter the composition of protistan community. An application to samples with a high abundance of metazoans showed that the blocking primer can reduce 42.1–72.4% of metazoan sequences, resulting the retrieval of a higher protistan richness and diversity. The use of the blocking primer helped to identify protistan community composition from a large size fraction, which is usually dominated by metazoans. Protistan community from the small (0.2–10 μm) and large (> 10 μm) size fractions exhibited a low similarity of 36.6% and shared 14.7% OTUs. About 63.8% of the OTUs were unique to the large fraction. Species from groups, such as Lepidodinium, Warnowia, Kareniaceae, Torodiniales, Phaeocystis, Chrysochromulina and Chrysophyceae, were enriched in the large fraction, indicating that they could be largely underestimated in studies that exclude the large-sized cells. Blocking primers are a promising tool to increase the efficiency for the characterization of protistan diversity in aquatic planktonic ecosystems.
... A number of modifications were suggested to inactivate oligonucleotides, including 3′-phosphate group [11], and chemically reversed 3′-terminal nucleotide (3′ to 5′)/inverted end [12] peptide nucleic acids (PNAs), locked nucleic acids (LNAs) etc. Such modified oligonucleotides may compete with the undesirable amplification primers for annealing site or prevent elongation by binding onto the target fragment between the two amplification primers [13][14][15][16][17][18][19]. However, these techniques can be implemented only for suppression of external primers before proceeding to internal primers during nested PCR. ...
Background
Recently we proposed efficient method to exclude undesirable primers at any stage of amplification reaction, here termed NOPE (NOnsense-mediated Primer Exclusion). According to this method, added oligonucleotide overlapping with the 3′-end of unwanted amplification primer (NOPE oligo) simultaneously provides a template for its elongation. This elongation disrupts specificity of unwanted primer, preventing its further participation in PCR. The suggested approach allows to rationally manage the course of PCR reactions in order to facilitate analysis of complex DNA mixtures as well as to perform multistage PCR bypassing intermediate purification steps.
Results
Here we apply NOPE method to DNA library preparation for the high-throughput sequencing (HTS) with the PCR-based introduction of unique molecular identifiers (UMI). We show that NOPE oligo efficiently neutralizes UMI-containing oligonucleotides after introduction of UMI into sample DNA molecules, thus allowing to proceed with further amplification steps without purification and associated loss of starting material. At the same time, NOPE oligo does not affect the efficiency of target PCR amplification.
Conclusion
We describe a simple, robust and cheap modification of UMI-labeled HTS libraries preparation procedure, that allows to bypass purification step and thus to preserve starting material which may be limited, e.g. circulating tumor DNA, circulating fetal DNA, or small amounts of isolated cells of interest. Furthermore, demonstrated simplicity and robustness of NOPE method should make it popular in various PCR protocols.
... The DNA degradation can be caused, for example, by heating treatments [59] and exposure to pH changes [60]. Furthermore different inhibitors of the Taq polymerase enzyme such as proteins, fats, polysaccharides, polyphenols, and other compounds that may be present in DNA may inhibit the DNA template amplification [61]- [64]. To manage these problems, the traceability of several selected DNA fragments differing in length, mapping on the inserted cassette, was studied along a complete industrial soybean processing chain ( Figure 6). ...
In this paper real-time, label free sensing principle based on Surface Plasmon Resonance (SPR) and gravimetric, i.e. Quartz Crystal Microbalance (QCM) are described and some applications to food analysis problems are reported. Affinity receptors are introduced and examples using antibodies, nucleic acid probes and biomimetic receptors i.e. molecular imprinted polymers (MIP) reported. In particualr pesticide, endotoxins, pathogens, Genetically Modified Organism (GMOs) detection and food origin analysis are reviewed.
... − Exploiting other methods with PCR hybridisation such as ligationdependent probe amplification (LPA), whose principle does not consist on amplifying the target sequence, but is rather based on the amplification of products resulting from the ligation of bipartite hybridisation probes [46,47]. Peptide nucleic acid (PNA)-mediated PCR clamping represents a rapid and effective means of assessing band identity [48,49]. ...
... The underlying principle of this approach is that the amount of DNA remaining in processed foodstuffs is sufficient for analytical purposes. The capacity to identify the genetic components of foodstuffs has been exploited to obtain traceability (Carcea et al., 2009;Pafundo, Gull ı, & Marmiroli, 2010;Samson, Gull ı, & Marmiroli, 2010;Turci et al., 2010), and to detect the presence of both genetically modified organisms (Bordoni et al., 2004;Marmiroli et al., 2008;Peano, Bordoni, et al., 2005;Peano, Lesignoli, et al., 2005) and allergens (European Patent n. 10154688.5; Pafundo, Gull ı, & Marmiroli, 2009;Pafundo, Gull ı, et al., 2010). ...
Fraudulent practices damage the market for premium olive oil; these can involve blending premium oil with oil produced from poor quality fruit, or adulteration with other plant oils. Methods based on assaying the DNA present in the oil are developing into a workable analytical tool. This review describes the use of “Food Genomics” to identify the varietal composition of olive oils, to ensure their conformity with legislation. The whole procedure is discussed, in the context of assembling an analytical platform suitable for the elaboration of an “identity card” for premium olive oils.
... Vestheim & Jarman (2008) found that a 10-fold excess of spacer-C3-CPG-blocking oligonucleotide compared with PCR primers was sufficient to reduce dominant predator amplicons from 100% to as low as 2.2% and even further at higher concentrations. The approach has been found to be efficient when designed to overlap with the primer-binding site, thus competitively preventing PCR primers from annealing [von Wintzingerode et al. 1997;Peano et al. ...
The analysis of food webs and their dynamics facilitates understanding of the mechanistic processes behind community ecology and ecosystem functions. Having accurate techniques for determining dietary ranges and components is critical for this endeavour. While visual analyses and early molecular approaches are highly labour intensive and often lack resolution, recent DNA-based approaches potentially provide more accurate methods for dietary studies. A suite of approaches have been used based on the identification of consumed species by characterization of DNA present in gut or faecal samples. In one approach, a standardized DNA region (DNA barcode) is PCR amplified, amplicons are sequenced and then compared to a reference database for identification. Initially, this involved sequencing clones from PCR products, and studies were limited in scale because of the costs and effort required. The recent development of next generation sequencing (NGS) has made this approach much more powerful, by allowing the direct characterization of dozens of samples with several thousand sequences per PCR product, and has the potential to reveal many consumed species simultaneously (DNA metabarcoding). Continual improvement of NGS technologies, on-going decreases in costs and current massive expansion of reference databases make this approach promising. Here we review the power and pitfalls of NGS diet methods. We present the critical factors to take into account when choosing or designing a suitable barcode. Then, we consider both technical and analytical aspects of NGS diet studies. Finally, we discuss the validation of data accuracy including the viability of producing quantitative data.
... 14 By adding a PNA able to bind next to the primer site on the target DNA, the specific inhibition of the PCR product, as revealed by agarose gel analysis, was obtained, allowing qualitative and semi-quantitative determination of the GMO presence. 21 This method is particularly suitable when no other equipment except the simple and cheap gel electrophoresis apparatus is available. ...
This tutorial review will address the issue of DNA determination in food by using Peptide Nucleic Acid (PNA) probes with different technological platforms, with a particular emphasis on the applications devoted to food authentication. After an introduction aimed at describing PNAs structure, binding properties and their use as genetic probes, the review will then focus specifically on the use of PNAs in the field of food analysis. In particular, the following issues will be considered: detection of genetically modified organisms (GMOs), of hidden allergens, of microbial pathogens and determination of ingredient authenticity. Finally, the future perspectives for the use of PNAs in food analysis will be briefly discussed according to the most recent developments.
... Specific PNA fragments were designed that inhibit PCR either by competing with the primer for annealing to target sequences, hybridizing adjacent and downstream of the primer, or hybridizing somewhere in the middle of the amplified sequence. The observed PCR inhibition by PNA reveals the particular GMO-related sequence in the sample and provides semiquantitative estimation of the GMO content [2]. A multiplex PCR followed by capillary gel electrophoresis (CGE) with laser-induced fluorescence (LIF) detection was reported for the simultaneous detection of five transgenic maizes (Bt11, T25, Mon810, GA21 and Bt176) and a reference gene (zein) in a single run. ...
Progress in genetic engineering has led to the introduction of genetically modified organisms (GMOs) whose genomes have been altered by the integration of a novel sequence conferring a new trait. To allow consumers an informed choice, many countries require food products to be labeled if the GMO content exceeds a certain threshold. Consequently, the development of analytical methods for GMO screening and quantification is of great interest. Exponential amplification by the polymerase chain reaction (PCR) remains a central step in molecular methods of GMO detection and quantification. In order to meet the challenge posed by the continuously increasing number of GMOs, various multiplex assays have been developed for the simultaneous amplification and/or detection of several GMOs. Classical agarose gel electrophoresis is being replaced by capillary electrophoresis (CE) systems, including CE chips, for the rapid and automatable separation of amplified fragments. Microtiter well-based hybridization assays allow high-throughput analysis of many samples in a single plate. Microarrays have been introduced in GMO screening as a technique for the simultaneous multianalyte detection of amplified sequences. Various types of biosensors, including surface plasmon resonance sensors, quartz crystal microbalance piezoelectric sensors, thin-film optical sensors, dry-reagent dipstick-type sensors and electrochemical sensors were introduced in GMO screening because they offer simplicity and lower cost. GMO quantification is performed by real-time PCR (rt-QPCR) and competitive PCR. New endogenous reference genes have been validated. rt-QPCR is the most widely used approach. Multiplexing is another trend in this field. Strategies for high-throughput multiplex competitive quantitative PCR have been reported.
While genetic manipulation for improvement of agricultural crops is not new, technologies such as gene editing that allow precise and targeted gene modifications are becoming more common. Highly specific, sensitive, and practical methods to detect such edits are desirable for a number of reasons. In this chapter, we provide readers with an overview of their application and what different technology platforms that can used to develop detection methods for edits. The platforms include PCR, digital PCR and sequencing and are discussed in the context of their strengths and weakness. This chapter also includes a discussion on challenges to differentiate a genetic change created through gene-editing technologies from same changes that might arise through other means such as conventional breeding or mutagenesis.
To standardize the rice-specific quantification methods, the criteria of six genes of rice (gos9, PLD, SPS, RBE4, ppi-PPF and oriazain) were compared and evaluated by ddPCR. The results revealed that SPS, RBE4 and ppi-PPF were single copy genes per haploid genome and species specificity and stable among different rice cultivars, by employing Lectin gene of soybean as internal reference gene. The established ddPCR systems were precise and reliable with an absolute LOQ of 10-20 copies/reaction. Furthermore, the robustness of these three assays was verified by performing an intra-laboratory repeatability validation and the results showed that the three endogenous genes of rice could be quantitated repeatedly and precisely above the LOQ. These ddPCR methods can reliably quantified the GM content even if the content was low to 0.1%, which were much more reliable than the results from real-time PCR using the same primers and probes.
Introduction DNA-Based Methodologies for GMO Analysis Strategies for Detection, Identification, and Quantification of GMOs Application of Current Methodologies to GMO Analysis References
This chapter discusses some examples of the application of polymerase chain reaction (PCR)-based approaches for the analysis of food toxicants. The use of PCR in assuring food traceability is an area of rapid expansion because the ability to monitor toxicants through the detection of trace amounts of DNA fragments overcomes many of the practicality and sensitivity limitations suffered by other technologies. Multiplex PCR represents a variant of PCR in which two or more DNA fragments are simultaneously amplified within a single reaction tube. This is achieved by including more than one primer pair to the reaction mixture. The approach is particularly relevant to food analysis where it is often necessary to test for the presence of a variety of toxicants in a single sample. In a nested PCR, two pairs of primers target a single locus. The first pair amplifies the target fragment in a conventional PCR reaction. The second pair anneals to sites within the first amplicon and amplifies an internal sequence. TaqMan assays are fluorogenic oligomers designed to hybridize to an internal site within the target sequence as defined by the conventional PCR primers.
Peptide nucleic acids (PNAs) are synthetic oligonucleotide analogues based on a pseudopeptide backbone that bind complementary DNA or RNA with high affinity and specificity. In this chapter, three PNA-based genotyping assays are described: PCR clamping, fluorescence-based recognition, and microarray platform. The first two methods are performed in solution, while the microarray method uses a solid surface.
Food safety and quality are very important issues receiving a lot of
attention in most countries by producers, consumers and regulatory and
control authorities. In particular, DNA analysis in food is becoming
popular not only in relation to genetically modified products (GMOs), in
which DNA modification is the "clue" of the novelty, but also in other
fields like microbiology and pathogen detection, which require long
times for the cultivation and specially in cases in which the
microorganisms are not cultivable like some viruses, as well as for
authenticity and allergen detection. A new topic concerning
"nutrigenetics and nutrigenomics" has also been mentioned, very
important but still in its infancy, which could lead in the future to a
personalized diet. In this chapter we have described the main areas of
food research and fields of application where DNA analysis is being
performed and the relative methods of detection, which are generally
based on PCR. The possibility/opportunity to detect DNA without previous
amplification (PCR-free) will be discussed. We have examined the
following areas: (1) genetically modified foods (GMOs); (2) food
allergens; (3) microbiological contaminations; (4) food authenticity;
(5) nutrigenetics/nutrigenomics.
In this study, four different protocols were tested for their ability to extract DNA from blended refined vegetable oils:
the in-house prepared Wizard and CTAB methods and the methods based on the use of the commercial kits Wizard® Magnetic DNA purification system for food and Nucleospin® for food. The performance of the extraction protocols was determined by end-point polymerase chain reaction (PCR) targeting
the soybean lectin gene with primers suitable for the amplification of small fragments and confirmed by real-time PCR with
specific hydrolysis probes. From the tested protocols, the Nucleospin method was the only one able to produce amplifiable
DNA from refined vegetable oils. To verify the presence of Roundup Ready® (RR) soybean, event-specific primers were used for end-point PCR assays. The amplification of trace amounts of RR soybean
by real-time PCR confirmed the label statements of two samples. The results highlight the importance of the DNA extraction
protocol and the critical choice of PCR primers on processed food matrices, such as refined oils. Considering the few reports
and difficulties pointed out in the literature to obtain amplifiable DNA from refined vegetable oils, the present results
can be a step forward in the traceability of refined oils regarding authenticity issues and genetically modified organism
detection.
KeywordsSoybean oil-Vegetable oil-DNA extraction-Real-time PCR-GMO detection-Refined oil
"Universal" or group-specific PCR primers have a tendency to predominately hybridise with the common sequences in samples with mixed templates. The result is that the rarer sequences are seldom retrieved by cloning or sequencing. The use of a blocking oligonucleotide (oligo) designed to specifically prevent amplification of dominant or unwanted DNA templates is an easy way to improve the amplification of rarer sequences. Here, we describe the different types of blocking principles and the different types of blocking oligos and give guidelines and examples of their application.
This paper reviews aspects relevant to detection and quantification of genetically modified (GM) material within the feed/food chain. The GM crop regulatory framework at the international level is evaluated with reference to traceability and labelling. Current analytical methods for the detection, identification, and quantification of transgenic DNA in food and feed are reviewed. These methods include quantitative real-time PCR, multiplex PCR, and multiplex real-time PCR. Particular attention is paid to methods able to identify multiple GM events in a single reaction and to the development of microdevices and microsensors, though they have not been fully validated for application.
A novel analytical procedure based on the combination of multiplex PCR, restriction analysis, and CGE-LIF to unambiguosly and simultaneously confirm the presence of multiple lines of genetically modified corn is proposed. This methodology is based on the amplification of event-specific DNA regions by multiplex PCR using 6-FAM-labeled primers. Subsequently, PCR products are digested by a mixture containing specific restriction endonucleases. Thus, restriction endonucleases selectively recognize DNA target sequences contained in the PCR products and cleave the double-stranded DNA at a given cleavage site. Next, the restriction digest is analyzed by CGE-LIF corroborating the length of the expected restriction fragments, confirming (or not) the existence of GMOs. For accurate size determination of the DNA fragments by CGE-LIF a special standard DNA mixture was produced in this laboratory for calibration. The suitability of this mixture for size determination of labeled DNA fragments is also demonstrated. The usefulness of the proposed methodology is demonstrated through the simultaneous detection and confirmatory analysis of samples containing 0.5% of GA21 and MON863 maize plus an endogenous gene of maize as control.
The Universal DNA microarray approach has been proposed for setting up several different DNA analysis. Such an approach is based on an arrayed set of oligonucleotides (the zipcode sequences) having common thermodinamic behaviour but being unrelated to any specific DNA sequence under study. This procedure, that can be combined to ligation, extension or minisequencing, requires the design of a set of probes specific for each target sequence (mutations, polymorphism, etc.) appended with complementary ZipCode (cZipCode) sequences. These probes are reacted against a proper target in solution in a multiplexed format using the above mentioned enzymatic reactions. The resulting products, bearing their own cZipCode are addressed to the location on the microarray where the corresponding ZipCode sequence has been spotted. Such an array is therefore "Universal" being unrelated to a specific molecular analysis. Here we present the set up and optimization of this procedure and its application to bacterial discrimination. Four presentations follow demonstrating the flexibility of the Universal array format in detecting pathogens and GMOs, monitoring bacterial diversity and typing SNPs.
A polyamide nucleic acid (PNA) was designed by detaching the deoxyribose phosphate backbone of DNA in a computer model and
replacing it with an achiral polyamide backbone. On the basis of this model, oligomers consisting of thymine-linked aminoethylglycyl
units were prepared. These oligomers recognize their complementary target in double-stranded DNA by strand displacement. The
displacement is made possible by the extraordinarily high stability of the PNA-DNA hybrids. The results show that the backbone
of DNA can be replaced by a polyamide, with the resulting oligomer retaining base-specific hybridization.
A new approach to rapid sequence comparison, basic local alignment search tool (BLAST), directly approximates alignments that optimize a measure of local similarity, the maximal segment pair (MSP) score. Recent mathematical results on the stochastic properties of MSP scores allow an analysis of the performance of this method as well as the statistical significance of alignments it generates. The basic algorithm is simple and robust; it can be implemented in a number of ways and applied in a variety of contexts including straightforward DNA and protein sequence database searches, motif searches, gene identification searches, and in the analysis of multiple regions of similarity in long DNA sequences. In addition to its flexibility and tractability to mathematical analysis, BLAST is an order of magnitude faster than existing sequence comparison tools of comparable sensitivity.
A novel method that allows direct analysis of single base mutation by the polymerase chain reaction (PCR) is described. The
method utilizes the finding that PNAs (peptide nucleic acids) recognize and bind to their complementary nucleic acid sequences
with higher thermal stability and specificity than the corresponding deoxyribooligonucleotides and that they cannot function
as primers for DNA polymerases. We show that a PNA/DNA complex can effectively block the formation of a PCR product when the
PNA is targeted against one of the PCR primer sites. Furthermore, we demonstrate that this blockage allows selective amplification/suppression
of target sequences that differ by only one base pair. Finally we show that PNAs can be designed in such a way that blockage
can be accomplished when the PNA target sequence is located between the PCR primers.
An empirical formula for thermal stability (Tm) prediction of PNA/DNA duplexes has been derived. The model is based on the Tm as calculated for the corresponding DNA/DNA duplex employing a nearest neighbour approach, by including terms for the pyrimidine
content and length of the PNA to take into account the increased thermostability of PNA/DNA hybrids and the asymmetry of the
PNA-DNA heteroduplex. The predictive power of the Tm prediction formula was challenged with an independent data set not used for model building. The Tm of >90% of the sequences was predicted within 5 K; 98% of the predicted Tms differ by not more than 10 K from the experimentally determined Tm.
An efficient, PCR based method for the selective amplification of DNA target sequences that differs by a single base pair is described. The method utilises the high affinity and specificity of PNA for their complementary nucleic acids and that PNA cannot function as primers for DNA polymerases.
Peptide Nucleic Acid (PNA) is a powerful new biomolecular tool with a wide range of important applications. PNA mimics the behaviour of DNA and binds complementary nucleic acid strands. The unique chemical, physical and biological properties of PNA have been exploited to produce powerful biomolecular tools, antisense and antigene agents, molecular probes and biosensors.
The PCR method has proved to be an invaluable tool for the specific and sensitive detection of genetically modified material (e.g., Roundup Ready Soybean and Bt-176 "Maximizer" Maize) in foodstuffs. The first step in the procedure, namely the purification of nucleic acids from the sample, is often the deciding factor in the production of meaningful results. In this study, we present two procedures that enable an efficient isolation of trace amounts of genetic material from both raw and highly processed foodstuffs. We show that for optimal, PCR-ready DNA purification from highly processed foodstuffs and PCR inhibitor-rich substances--such as cocoa-containing products--adapted protocols for the QIAGEN QIAamp DNA Stool Mini Kit can be utilized. For complete DNA isolation from raw foodstuffs, a protocol using the DNeasy Plant Mini Kit is presented.
The astonishing discovery that peptide nucleic acids (PNAs, B=nucleobase), in spite of their drastic structural difference to natural DNA, are better nucleic acid mimetics than many other oligonucleotides has resulted in an explosion of research into this class of compounds. The synthesis, physical properties, and biological interactions of PNAs as well as their chimeras with DNA and RNA are summarized here.
Since the investigation of oligonucleotides as potential therapeutics that target nucleic acids was initiated, the search for nucleic acid mimetics with improved properties, such as strengthened binding-affinity to complementary nucleic acids, increased biological stability, and improved cellular uptake, has accelerated rapidly. In 1991 Nielsen et al. first described what is undoubtedly one of the most interesting of the new derivatives, the polyamide or peptide nucleic acids (PNAs), in which the entire sugar-phoshate backbone is replaced by an N-(2-aminoethyl)glycine polyamide structure. Since even minor structural changes in oligonucleotides, such as the replacement of an oxygen atom by sulfur (phosphorothioates), or by a neutral methyl group (methyl phosphonates), result in a decrease in binding affinity, it was even more astonishing to find that the drastic structural changes in PNAs result in nucleic acid mimetics with higher binding-affinity to complementary DNA and RNA than unmodified oligonucleotides. The remarkable binding properties of PNAs have spawned a rapidly expanding new field of research, where the targets are the synthesis of PNAs and PNA analogues, and their application as therapeutics, DNA diagnostics, and tools in biotechnology. In addition, investigation of PNAs and PNA/DNA chimeras can be used to generate information on the structural and biological properties of DNA and RNA themselves. Furthermore, they may trigger the generation of new ideas on models for alternative living systems and potential transitions between different genetic systems.
Peptide nucleic acids (PNAs) are oligonucleotide mimics containing a pseudopeptide chain, which are able to bind complementary DNA tracts with high affinity and selectivity. Two mixed-sequence PNA undecamers (1 and 2) were synthesized and their double-stranded adducts with the complementary oligonucleotides (3 and 4) were revealed by the appearance of the corresponding peak in anion-exchange HPLC. A DEAE column was used and elution was performed with aqueous Tris buffer (pH 8) and an ionic strength gradient (0–0.5 M NaCl). The same effect was not observed with non-complementary oligonucleotides. The stability of the PNA–DNA adducts under the conditions used in the chromatographic system was studied as a function of temperature. Furthermore, in competition experiments double-stranded oligonucleotides were challenged by a PNA complementary to one strand: the formation of the PNA–DNA hybrid and the displacement of the non-complementary strand were observed with high specificity. The results suggest a possible use of ion-exchange HPLC for studying PNA–DNA interactions, and indicate the efficiency of PNA probes in the chromatographic analysis of DNA.
The principle of direct detection of recombinant DNA in food by the polymerase chain reaction (PCR) is discussed following the three main steps: DNA-extraction, amplification by PCR and verification of PCR products.Suitable methods for genomic DNA isolation from homogenous, heterogeneous, low DNA containing matrices (e.g. lecithin), gelatinising material (e.g. starch), derivatives and finished products based on classical protocols and/or a combination with commercially available extraction kits are discussed. Various factors contribute to the degradation of DNA such as hydrolysis due to prolonged heat-treatment, nuclease activity and increased depurination and hydrolysis at low pH. The term “DNA quality” is defined as the degree of degradation of DNA (fragment size less than 400 bp in highly processed food) and by the presence or absence of potent inhibitors of the PCR and is, therefore, a key criterion. In general, no DNA is detectable in highly heat-treated food products, hydrolysed plant proteins (e.g. soya sauce), purified lecithin, starch derivatives (e.g. maltodextrins, glucose syrup) and defined chemical substances such as refined soya oil.If the nucleotide sequence of a target gene or stretch of transgenic DNA is already known specific primers can be synthesised and the segment of rDNA amplified. Detection limits are in the range 20 pg–10 ng target DNA and 0.0001–1% mass fraction of GMO. Amplification products are then separated by agarose gel electrophoresis and the expected fragment size estimated by comparison with a DNA molecular weight marker.Several methods are used to verify PCR results and they vary in reliability, precision and cost. They include specific cleavage of the amplification products by restriction endonucleases or the more time-consuming, but also more specific, transfer of separated PCR-products onto membranes (Southern Blot) followed by hybridisation with a DNA probe specific for the target sequence. Alternatively, PCR products may be verified by direct sequencing. Nested-PCR assays combines high specificity and sensitivity.Methods for the screening of 35S-promoter, NOS-terminator and other marker genes used in a wide range of GMOs, the specific detection of approved products such as FlavrSavr™ tomatoes, Roundup Ready™ Soya, Bt-maize 176 and official validated methods for potatoes and genetically modified micro-organisms, that have a model character, are available. Methods to analyse new GMO products are being validated by interlaboratory tests and new techniques are in development (e.g. EC project: DMIF-GEN). However, these efforts may be hampered by the lack of availability of GMO reference material as well as specific sequence information which so far can only be obtained from the suppliers.
Two different PCR-based approaches for the quantitative analysis of genetically modified organism (GMO) – components in foods are presented using Soybean derived samples as an example. The first method – a double competitive PCR – is well suited to determine threshold levels of GMO content in food. The other – PCR on-line measurement – is suited to determine ratios of transgenic versus non-transgenic component. Both methods provide a means to alleviate the problems of standardisation encountered with simple qualitative PCR approaches and will allow to cope with threshold levels for GMO, once issued by legislative bodies.
Validated analytical methods are necessary in order to show compliance with labelling concerning conventionally produced food or food containing genetically modified organisms (GMOs). Methods to be used in arbitration should be collaboratively tested according to standardisation protocols. According to the Council Regulation of 1998, one of the marker components being the criterion for labelling of products from soy beans and maize is the genetically altered DNA. A qualitative polymerase chain reaction method which has recently been validated in a collaborative trial can be used for initial screening of food products in order to detect GMOs. Furthermore, this screening method has to be validated for processed food. In addition, there is a strong need for quantitative methods with a view to a threshold value for labelling and to distinguish between the source of GMOs in food products (ingredients or additives).
DNA analogues are currently being intensely investigated owing to their potential as gene-targeted drugs. Furthermore, their properties and interaction with DNA and RNA could provide a better understanding of the structural features of natural DNA that determine its unique chemical, biological and genetic properties. We recently designed a DNA analogue, PNA, in which the backbone is structurally homomorphous with the deoxyribose backbone and consists of N-(2-aminoethyl)glycine units to which the nucleobases are attached. We showed that PNA oligomers containing solely thymine and cytosine can hybridize to complementary oligonucleotides, presumably by forming Watson-Crick-Hoogsteen (PNA)2-DNA triplexes, which are much more stable than the corresponding DNA-DNA duplexes, and bind to double-stranded DNA by strand displacement. We report here that PNA containing all four natural nucleobases hybridizes to complementary oligonucleotides obeying the Watson-Crick base-pairing rules, and thus is a true DNA mimic in terms of base-pair recognition.
Hereditary hemochromatosis (HH), an iron overload disease, is the most common known inheritable disease. The most prevalent form of HH is believed to be the result of a single base-pair mutation. We describe a rapid homogeneous mutation analysis method that does not require post-polymerase chain reaction (PCR) manipulations. This method is a marriage of three emerging technologies: rapid cycling PCR thermal cyclers, peptide nucleic acid (PNA) probes, and a new double-stranded DNA-selective fluorescent dye, Sybr Green I. The LightCycler is a rapid thermal cycler that fluorometrically monitors real-time formation of amplicon with Sybr Green I. PNAs are DNA mimics that are more sensitive to mismatches than DNA probes, and will not serve as primers for DNA polymerases. PNA probes were designed to compete with PCR primers hybridizing to the HH mutation site. Fully complemented PNA probes at an 18:1 ratio over DNA primers with a mismatch result in suppression of amplicon formation. Conversely, PNA probes with a mismatch will not impair the binding of a complementary primer, culminating in amplicon formation. A LightCycler-based rapid genetic assay has been developed to distinguish HH patients from HH carriers and normal individuals using PNA clamping technology.
PNA (peptide nucleic acid) is a DNA mimic with a pseudopeptide (polyamide) backbone which can be used to target double stranded DNA with high sequence specificity. PNA therefore has great potential in the development of biomolecular tools for manipulation of DNA as well as for the development of DNA targeted gene therapeutic drugs. The status of this field is discussed in terms of PNA binding modes and mechanism as well as applications.
Concerns have been raised about the potential effects of transgenic introductions on the genetic diversity of crop landraces and wild relatives in areas of crop origin and diversification, as this diversity is considered essential for global food security. Direct effects on non-target species, and the possibility of unintentionally transferring traits of ecological relevance onto landraces and wild relatives have also been sources of concern. The degree of genetic connectivity between industrial crops and their progenitors in landraces and wild relatives is a principal determinant of the evolutionary history of crops and agroecosystems throughout the world. Recent introductions of transgenic DNA constructs into agricultural fields provide unique markers to measure such connectivity. For these reasons, the detection of transgenic DNA in crop landraces is of critical importance. Here we report the presence of introgressed transgenic DNA constructs in native maize landraces grown in remote mountains in Oaxaca, Mexico, part of the Mesoamerican centre of origin and diversification of this crop.
Biospecific interaction analysis (BIA) was performed using surface plasmon resonance (SPR) and biosensor technologies to detect genetically modified Roundup Ready soybean gene sequences. We first immobilized, on SA sensor chips, single-stranded biotinylated oligonucleotides containing soybean lectin and Roundup Ready gene sequences, and the efficiency of hybridization to oligonucleotide probes differing in length was determined. Second, we immobilized biotinylated PCR products from nontransgenic soybeans (genomes carrying only the lectin gene), as well as from genetically modified Roundup Ready soybean, and we injected the oligonucleotide probes. Furthermore, we used the sensor chips carrying either lectin and Roundup Ready soybean PCR products or 21-mer oligonucleotide as probes, and we injected both nonpurified and purified asymmetric PCR products. The results obtained show that 13 and 15 mer oligonucleotides are suitable probes to detect genetically modified Roundup Ready soybean gene sequences (either target oligonucleotides or PCR products) under standard BIA experimental conditions. By contrast, when 11 mer DNA probes were employed, no efficient hybridization was obtained. All the SPR-based formats were found to be useful for detection of Roundup Ready gene sequences, suggesting that these procedures are useful for the real-time monitoring of hybridization between target single-stranded PCR products, obtained by using as substrates DNA isolated from normal or transgenic soybeans, and oligonucleotide or PCR-generated probes, therefore enabling a one-step, nonradioactive protocol to perform detection.
Two previously developed platforms, a multiplex polymerase chain reaction (PCR) and a peptide nucleic acid (PNA) array, the former allowing for the simultaneous detection of five transgenes and two endogenous controls in food and feed matrices and the latter for the assessment of the identity of amplified PCR products, were combined in order to develop a PNA array device for the screening of genetically modified organisms (GMOs) in food. PNA probes were opportunely designed, synthesized, and deposited on commercial slides. The length of the probes as well as the distance of the probes from the surface were evaluated and found to be critical points. The most suitable probes were found to be 15-mer PNAs linked to the slide surface by means of two 2-(2-aminoethoxy)ethoxyacetic acids as spacers. The device was tested on a model system constituted by flour samples containing a mixture of standards at known concentrations of transgenic material, in particular Roundup Ready soybean and Bt11, Bt176, Mon810, and GA21 maize: The DNA was amplified using the specific multiplex PCR method and tested on the PNA array. The method proposed was found to be able to correctly identify every GMO present in the tested samples.
Development of a PNA array platform for the detection of GMOs in food
- A Germini
- S Rossi
- A Zanetti
- R Corradini
- C Fogher
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The European Parliament and the Council of the European Union on genetically modiWed food and feed
Regulation (EC) No 1829/2003 The European Parliament
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Basic local alignment search tool
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