scholarly journals Challenges for the characterization of genetically modified animals by the qPCR technique in the era of genomic editing

2021 ◽  
Vol 3 (1) ◽  
Author(s):  
Ribrio Ivan Tavares Pereira Batista ◽  
Dárcio Ítalo Alves Teixeira ◽  
Vicente José de Figueirêdo Freitas ◽  
Luciana Magalhães Melo ◽  
Joanna Maria Gonçalves Souza-Fabjan
Keyword(s):  
Copy Number ◽  
Genetically Modified Organisms ◽  
Genetically Modified ◽  
Transgenic Animals ◽  
Real Time Quantitative Pcr ◽  
Transgenic Organisms ◽  
Pcr Technology ◽  
Genomic Editing

Characterization of genetically modified organisms through determination of zygosity and transgene integration concerning both copy number and genome site is important for breeding a transgenic line and the use of these organisms in the purpose for which it was obtained. Southern-blot, fluorescence in situ hybridization or mating are demanding and time-consuming techniques traditionally used in the characterization of transgenic organisms and, with the exception of mating, give ambiguous results. With the emergence of the real-time quantitative PCR technology, different applications have been described for the analysis of transgenic organisms by determination of several parameters to transgenic analysis. However, the accuracy in quantitation by this method can be influenced in all steps of quantification. This review focuses on the aspects that influence pre-analytical steps (DNA extraction and DNA quantification methods), quantification strategies and data analysis in quantification of copy number and zygosity in transgenic animals.

scholarly journals Determination of the Absolute Number of Transgene Copies in CMVFUT Transgenic Pigs

2012 ◽  
Vol 12 (3) ◽  
pp. 349-356 ◽  
Author(s):  
Daniel Lipiński ◽  
Joanna Zeyland ◽  
Andrzej Pławski ◽  
Ryszard Słomski
Keyword(s):  
Copy Number ◽  
Transgenic Animals ◽  
Promoter Sequence ◽  
Absolute Number ◽  
Transgenic Pigs ◽  
Cmv Promoter ◽  
Genetic Construct ◽  
Coding Sequence ◽  
The Absolute

Determination of the Absolute Number of Transgene Copies in CMVFUT Transgenic PigsThe aim of this research was to determine the number of transgene copies in the DNA of transgenic pigs. The copy number of the transgene was analysed in the transgenic animals with introduced pCMVFUT genetic construct containing a coding sequence of human H transferase under a control of CMV promoter. The copy number of the transgene that had integrated with the genome of the transgenic animals was analysed by qPCR with SYBR Green dye, which enabled nonspecific double-stranded DNA detection. CMVFT-2F and CMVFT-2R primers were used to amplify a 149 bp fragment of DNA. Forward primer had a sequence complementary to a promoter sequence and reverse primer to a coding sequence of H transferase. The copy number of the transgene in the examined samples was established by plotting the CT values obtained on a standard curve, which had been set by the usage of the CT values for the successive standard dilutions with known copy number (1.438-1.431 copies). As a standard we used pCMVFut genetic construct hydrolyzed with Not I restriction enzyme to a linear form. The real-time PCR results helped to establish the range of 3 - 4 as the number of the transgene copies that had integrated to the swine genome.

scholarly journals Calculation of Measurement Uncertainty in Quantitative Analysis of Genetically Modified Organisms Using Intermediate PrecisionA Practical Approach

2007 ◽  
Vol 90 (2) ◽  
pp. 582-586 ◽  
Author(s):  
Jana Žel ◽  
Kristina Gruden ◽  
Katarina Cankar ◽  
Dejan tebih ◽  
Andrej Blejec
Keyword(s):  
Nucleic Acids ◽  
Measurement Uncertainty ◽  
Genetically Modified Organisms ◽  
Genetically Modified ◽  
Laboratory Diagnostics ◽  
Quantitative Characterization ◽  
Intermediate Precision ◽  
Food And Feed ◽  
Repeatability And Reproducibility

Abstract Quantitative characterization of nucleic acids is becoming a frequently used method in routine analysis of biological samples, one use being the detection of genetically modified organisms (GMOs). Measurement uncertainty is an important factor to be considered in these analyses, especially where precise thresholds are set in regulations. Intermediate precision, defined as a measure between repeatability and reproducibility, is a parameter describing the real situation in laboratories dealing with quantitative aspects of molecular biology methods. In this paper, we describe the top-down approach to calculating measurement uncertainty, using intermediate precision, in routine GMO testing of food and feed samples. We illustrate its practicability in defining compliance of results with regulations. The method described is also applicable to other molecular methods for a variety of laboratory diagnostics where quantitative characterization of nucleic acids is needed.

Determination of Knowledge, Attitude, Behavior about Genetically Modified Organisms in Nursing School Students

2013 ◽  
Vol 55 (4) ◽  
pp. 297 ◽  
Author(s):  
turker turker ◽  
necmettin kocak ◽  
Ibrahim Aydin ◽  
hakan istanbulluoglu ◽  
nuri yildiran ◽  
...  
Keyword(s):  
Genetically Modified Organisms ◽  
Genetically Modified ◽  
Nursing School ◽  
School Students ◽  
Attitude Behavior

Development of sampling approaches for the determination of the presence of genetically modified organisms at the field level

2010 ◽  
Vol 396 (6) ◽  
pp. 2031-2041 ◽  
Author(s):  
Jelka Šuštar-Vozlič ◽  
Katja Rostohar ◽  
Andrej Blejec ◽  
Petra Kozjak ◽  
Zoran Čergan ◽  
...  
Keyword(s):  
Genetically Modified Organisms ◽  
Genetically Modified ◽  
Field Level

scholarly journals UNDERSTANDING OF GENETICALLY MODIFIED ORGANISMS

2012 ◽  
Vol 2 (2) ◽  
pp. 61-66
Author(s):  
Željko Kaluđerović ◽  
◽  
Jovana Potpara ◽  
Keyword(s):  
Genetic Engineering ◽  
Genetically Modified Organisms ◽  
Genetically Modified ◽  
New Technology ◽  
A Priori ◽  
Point Of View ◽  
Individual Species ◽  
Transgenic Organisms ◽  
Ethical Point ◽  
Special Challenge

During the last sixteen years biotechnology, genetic engineering, transgenic organisms or genetically modified organisms (GMOs) have been raising numerous controversies. In the scientific sphere, genetic engineering and GMOs represent a special challenge for geneticists, breeders and physicians, in philosophy it is a topic of interest for bioethicists and agricultural ethicists, environmentalists are interested in the interconnectictions between new technology and environment protection, for multinational companies this is a potential source of huge profits, and for certain governments they represent an instrument for strategic control of food production within their countries as well as internationally. By taking into account the views of both advocates and opponents of this "revolutionary" method, authors believe that we should not a priori reject new and insufficiently studied technologies, but that in this particular it is necessary to be extremely cautious, in other words that from (bio)ethical point of view only those GMO investigations limited to scientific purposes are justified, provided that all required precautions have been taken. Also, authors are of the opinion that in this region as well as in Europe as a whole, at this moment, transgenic organisms are not necessery, neither in agricultural production nor in the food chain. Arguments for such a statement are found primarily in the potential issues that intentional breeding of GMOs might inflict upon the human health and environment. Namely, if borders of individual species are not overstepped and if their endogenous traits are made stronger, the potential risk of causing irreparable damage for both present and future generations which may be brought by changed biological succession will be reduced, i.e. one of the four fundamental bioethical principles will be applied and that is the nonmaleficence. Further intentional decreasing of biodiversity should not be allowed, which means that it is necessary to respect as much as it is possible the complexity of the very nature, its autonomy and "otherness".

scholarly journals Characterization of Genetically Modified Maize in Weakly Contaminated Seed Batches and Identification of the Origin of the Adventitious Contamination

2007 ◽  
Vol 90 (4) ◽  
pp. 1098-1106 ◽  
Author(s):  
Laetitia Petit ◽  
Galle Pagny ◽  
Fabienne Baraige ◽  
Anne-Ccile Nignol ◽  
David Zhang ◽  
...  
Keyword(s):  
Ssr Markers ◽  
Genetically Modified Organisms ◽  
Genetically Modified ◽  
The European Union ◽  
Maize Seeds ◽  
Genetic Modifications ◽  
Polymerase Chain ◽  
The Individual ◽  
The Eu

Abstract So far, relatively few genetically modified plants (GMPs) have been planted in the European Union (EU). However, in France, seed batches weakly contaminated by unidentified GM materials have recently been detected among commercial maize seeds (14 seed batches positive out of 447 analyzed). We have developed a 3-step approach to precisely identify the genetic modifications detected in such maize seed batches. First, to isolate GMPs derived from the contaminated seed batches, 10 000 maize seeds of each batch were planted and screened by polymerase chain reaction (PCR) on 100-plant batches, then on 10-plant subbatches, and finally, plant by plant. In a second step, specific identification of the individual GMPs was performed. Finally, to determine the origin of the contamination, each individual GMP was analyzed by simple sequence repeat (SSR) markers. The results showed that all batches were contaminated by few GM seeds, having a GM content <0.1%. Finally, 12 individual GMPs have been isolated from 17 plant pools that were tested positive either for P35-S and/or T-Nos. MON810 and T25 transformation events approved for cultivation in the EU were detected in 7 individual GMPs. The other seed batches were contaminated by genetically modified organisms (GMOs) that are not approved in the EU, including GA21 or the stacking MON810/T25. Presumable identification of T14 was also achieved following sequencing of 1 individual GMP. The data also showed that most of the seed batches were contaminated by several transformation events. Finally, analysis of SSR markers indicated that the contaminations were essentially due to cross-pollination in the seed production process.

scholarly journals Production of Certified Reference Materials for the Detection of Genetically Modified Organisms

2002 ◽  
Vol 85 (3) ◽  
pp. 775-779 ◽  
Author(s):  
Stefanie Trapmann ◽  
Heinz Schimmel ◽  
Gerard Nico Kramer ◽  
Guy van den Eede ◽  
Jean Pauwels
Keyword(s):  
Reference Materials ◽  
Genetically Modified Organisms ◽  
Production Control ◽  
Certified Reference Materials ◽  
Genetically Modified ◽  
Base Material ◽  
Genetically Modified Organism ◽  
International Organization ◽  
Roundup Ready

Abstract Certified reference materials (CRMs) are an essential tool in the quality assurance of analytical measurements. They are produced, certified, and used in accordance with relevant ISO (International Organization for Standardization) and BCR (Community Bureau of Reference) guidelines. The Institute for Reference Materials and Measurements (IRMM; Geel, Belgium) has produced the first powdery genetically modified organism (GMO) CRMs in cooperation with the Institute for Health and Consumer Protection (Ispra, Italy). Until now, different weight percentages in the range of 0–5%for 4 GMOs in Europe were produced and certified: Bt (Bacillus thuringiensis)-11 and Bt-176 maize, Roundup Ready® soybean, and MON810 maize. Bt-11 and Bt-176 maize and Roundup Ready soybean were produced by IRMM on behalf of Fluka Chemie AG (Buchs, Switzerland). Characterization of used base material is the first step in production and is especially important for GMO CRMs. The production of powdery GMO CRMs and methods used for production control are described. Thorough control of homogeneity and stability are essential for certification of reference materials and ensure validity of the certificate for each bottle of a batch throughout a defined shelf-life. Because production of reference materials and their maintenance are very labor- and cost-intensive tasks, the usefulness of new types of GMO CRMs must be estimated carefully.

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