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Pairwise sequence alignment of the synthetic construct opAFP-GHc (AY594644.1) used to produce the transgenic Atlantic salmon and the related construct named opAFP-GHc2 (AY687640.1). The constructs differ on the 5'untranslated sequence (5'UTR) of the growth hormone (GH) cDNA.
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The detection of transgenic animals in food and environmental samples will require the development of reliable screening assays. The identification of junction regions between genetic elements is the unequivocal way to prove the presence of transgene constructs. However, a sample from the transgenic organism or genetic construct is not always avail...
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... also downloaded the sequence from a similar construct named opAFP-GHc2 (AY687640.1) associated with a patent application on the same subject (Hew & Fletcher, 2001;Hew, Fletcher, & Davies, 1995) (Figure 1). The alignment of the two constructs was carried out using the Muscle tool implemented in Geneious Pro software v10.0 (Biomatters Ltd., Auckland, New Zealand) (Edgar, 2004). ...
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... alignment of the opAFP-GHc and opAFP-GHc2 constructs revealed that they differ in the 5'-untranslated sequence (5'UTR) of the GH cDNA (Figure 1). ...
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... the opAFP-GHc construct (AY594644.1) is usually mentioned in the literature as being used for the production of the AquAdvantage salmon (Du, et al., 1992;Hew, et al., 1995). However, considering the possible use of opAFP-GHc2 in transgenic fishes, we avoided the design of PCR primers within the 5'UTR variable region (Figure 1). ...
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... pair combinations using ZameAFP2027F as a forward primer resulted in a slightly higher PCR efficiency compared to ZameAFP2073F. The amplification of the AFP-GH junction in a transgenic animal will yield a slightly larger amplicon due to the presence of a short ~80bp 5'UTR between the AFP and GH cDNA (Figure 1). Nevertheless, the amplified products will be shorter than 250bp and thus likely to amplify in degraded DNA samples. ...
Citations
... The success of the amplification was determined by electrophoresis using standard 1Á2% agarose gels. The amplified products were prepared and sequenced as described in Castro et al. (2017). The analysis of the resulting electropherograms and the de novo assembly was performed with the Geneious Prime 2019.2.3 (https://www.geneious.com). ...
The mitochondrial genome of Neurospora crassa has been less studied than its nuclear counterpart, yet it holds great potential for understanding the diversity and evolution of this important fungus. Here we describe a new mitochondrial DNA (mtDNA) complete sequence of a N. crassa wild type strain. The genome with 64,839 bp revealed 21 protein coding genes and several hypothetical open reading frames (ORFs) with no significant homology to any described gene. Five large repetitive regions were identified across the genome, including partial or complete genes. The largest repeated region holds a partial nd2 section that was also detected in Neurospora intermedia, suggesting a rearrangement that occurred before the N. crassa speciation. Interestingly, N. crassa has a palindrome adjacent to the partial nd2 repeated region possibly related with the genomic rearrangement, which is absent in N. intermedia. Finally, we compared the sequences of the three available N. crassa complete mtDNAs and found low levels of intraspecific variability. Most differences among strains were due to small indels in non‐coding regions. The revisiting of the N. crassa mtDNA forms the basis for future studies on mitochondrial genome organization and variability.
... Our poor knowledge on wildlife and microbes adds to this already long list of challenges. Concerning the specific field of GMOs (genetically modified organisms) detection, the new technologies of genome editing avoiding traceable insertion of exogenous sequences [20] such as CRISPR-Cas9 add extra problems to which solutions are still unseen and legislation not yet ready. ...
Forensic genetics is the application of genetics to human and nonhuman material for the resolution (and prevention) of legal conflicts. Nonhuman DNA applications are increasing, from the ancillary role in criminalistics to the control of protected species and their products, microbial identification in bioterrorism or medical malpractice. We review this growing applications’ scope and identify the major current difficulties, mainly resulting from the lack of standards and genetic databases as well as the poor or absent taxonomic definition of many major groups.
... If there is a potential for GM foods to become inadvertently mixed with non-GM foods, during marketing or processing, detection tools would be desirable to allow detection of GM salmon in food products. A PCR method (gel based) was proposed by Castro et al. (2017) for the detection of the AquAdvantage® salmon but not yet tested on real samples. ...
... A sequence of 1400 bp based on the transgenic insert sequence in AquAdvantage® salmon was synthesized and cloned in the pUC57 plasmid. We preferred the creation of a fragment by synthesis to prepare a positive control to the method proposed by Castro et al. (2017) and assembling DNA fragments mimicking junctions of transgenic elements present in the AquAdvantage® salmon as the creation of a plasmid by synthesis is easier and faster. The tests for limit of detection were thus performed on plasmid DNA and not on genomic DNA. ...
Genetic modifications (GM) have been applied to salmon to generate fast-growing strains for potential use in aquaculture. In November 2015, the first transgenic salmon (AquAdvantage® Atlantic salmon) was accepted for commercialization in the USA under defined conditions. The presence of GM food products in the marketplace stimulates the need for detection methods to allow screening for the presence of genetic modifications in seafood products. This paper first shows that it is possible to obtain amplifiable DNA from raw and processed products containing salmon. Detection methods by real-time PCR are proposed in this work. An endogenous gene target was designed to detect salmonid species DNA in samples. In addition, detection methods using real-time PCR were developed for two GM salmon possessing growth hormone transgenes: the AquAdvantage® Atlantic salmon (Salmo salar) developed by AquaBounty for commercial purposes, and the coho salmon (Oncorhynchus kisutch) developed for research purposes by Fisheries and Oceans Canada. The methods are able to detect at least 20 copies of the target. It was found however that one of the construct-specific methods for the AquAdvantage® salmon detection did not work on AquAdvantage® genomic DNA even though it works on the sequence published in GenBank. The other assay however was found to reliably detect AquAdvantage® transgenic sequences in genomic DNA.
... The first genetically modified animal (AquAdvantage salmon) is on the verge of being approved for human consumption in different countries [249]. New methods are being developed to detect the genetically modified salmon in food products [250,251]. Strong legislation is expected to regulate the presence of this transgenic animal in foods and environmental samples [252,253] and, consequently, reliable and sensitive methods for its detection will be required by regulatory and scientific agencies worldwide [245,254]. ...
While traditional forensic genetics has been oriented towards using human DNA in criminal investigation and civil court cases, it currently presents a much wider application range, including not only legal situations sensu stricto but also and, increasingly often, to preemptively avoid judicial processes. Despite some difficulties, current forensic genetics is progressively incorporating the analysis of nonhuman genetic material to a greater extent. The analysis of this material—including other animal species, plants, or microorganisms—is now broadly used, providing ancillary evidence in criminalistics in cases such as animal attacks, trafficking of species, bioterrorism and biocrimes, and identification of fraudulent food composition, among many others. Here, we explore how nonhuman forensic genetics is being revolutionized by the increasing variety of genetic markers, the establishment of faster, less error-burdened and cheaper sequencing technologies, and the emergence and improvement of models, methods, and bioinformatics facilities.
Genetically modified (GM) Atlantic salmon, AquAdvantage (AquAd), was the first GM animal approved officially for human consumption. Many countries monitor the use of this product under their GM regulations, but a pragmatic system for AquAd-specific detection is needed. Here, we developed a real-time polymerase chain reaction method with high sensitivity for detection of AquAd in foods. This method showed high specificity for the AquAd transgene and the detection limit was 12.5-25 targeted DNA copies per test reaction. An inter-laboratory study using the method developed demonstrated reproducibility at >0.1% (w/w) AquAd content.
