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Real-time quantification of wild-type contaminants in glyphosate tolerant soybean.

Battistini E, Noli E - BMC Biotechnol. (2009)

Bottom Line: The soybean wt sequence at the RR locus was characterized and found to be highly conserved among conventional genotypes, thus allowing the detection of possibly any soybean non-trait contaminant.In addition, results obtained from the analysis of wt-spiked RR samples demonstrate that it is possible to use the real-time PCR assay to quantify the non-trait contamination with an acceptable degree of accuracy.In principle this approach could be successfully applied to any transgenic event, provided that the wild-type sequence is conserved and single copy.

View Article: PubMed Central - HTML - PubMed

Affiliation: Seed Research and Testing Laboratory (LaRAS), Department of Agroenvironmental Sciences and Technologies (DiSTA), Alma Mater Studiorum University of Bologna, viale Fanin 40, Bologna 40127, Italy. ebattistini@agrsci.unibo.it

ABSTRACT

Background: Trait purity is a key factor for the successful utilization of biotech varieties and is currently assessed by analysis of individual seeds or plants. Here we propose a novel PCR-based approach to test trait purity that can be applied to bulk samples. To this aim the insertion site of a transgene is characterized and the corresponding sequence of the wild-type (wt) allele is used as diagnostic target for amplification. As a demonstration, we developed a real-time quantitative PCR method to test purity of glyphosate tolerant (Roundup Ready, RR) soybean.

Results: The soybean wt sequence at the RR locus was characterized and found to be highly conserved among conventional genotypes, thus allowing the detection of possibly any soybean non-trait contaminant. On the other hand, no amplification product was obtained from RR soybean varieties, indicating that the wt sequence is single copy and represents a suitable marker of conventional soybean presence. In addition, results obtained from the analysis of wt-spiked RR samples demonstrate that it is possible to use the real-time PCR assay to quantify the non-trait contamination with an acceptable degree of accuracy.

Conclusion: In principle this approach could be successfully applied to any transgenic event, provided that the wild-type sequence is conserved and single copy. The main advantages of the assay here described derive from its applicability to bulk samples, which would allow to increase the number of single seeds or plants forming the analytical sample, thus improving accuracy and throughput while containing costs. For these reasons this application of quantitative PCR could represent a useful tool in agricultural biotechnology.

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Schematic representation of the PCR-walking approach to isolate the wt allele at the RR locus. Top drawing: original integration site of the RR construct in conventional soybean. Steps (1–5) of PCR-walking. Only EcoRV fragment isolation procedure is shown.
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Figure 2: Schematic representation of the PCR-walking approach to isolate the wt allele at the RR locus. Top drawing: original integration site of the RR construct in conventional soybean. Steps (1–5) of PCR-walking. Only EcoRV fragment isolation procedure is shown.

Mentions: Genomic DNA extracted from a conventional soybean genotype was digested with ScaI and EcoRV for which the absence of restriction sites in the 3' genomic flanking sequence had been previously ascertained (Figure 2). DNA fragments were subsequently blunt-ligated with adapters and PCR-walking reactions were performed using forward adapter primers and reverse primers specific for the known sequence flanking the 3' end of the RR insert. PCR products obtained by the nested amplification of the adapter-fragments were purified and characterized, providing sequences for the ScaI/plant DNA and the EcoRV/plant DNA. By combining the two partially overlapping sequences, a 714 bp sequence characteristic of the non-GM genome at the RR insertion site was obtained (Figure 3). Of this, 84 bp corresponded to the flanking region at the 3' end previously reported, whereas the remaining newly described 630 bp represented the wt sequence upstream of the RR insertion site [20]. However, no homology with the plant DNA flanking the 5' end of the insert was found indicating that, following the integration of the transgene, major rearrangements had taken place [20]. In addition, no homology with other sequences in GenBank was detected by BLAST analysis.


Real-time quantification of wild-type contaminants in glyphosate tolerant soybean.

Battistini E, Noli E - BMC Biotechnol. (2009)

Schematic representation of the PCR-walking approach to isolate the wt allele at the RR locus. Top drawing: original integration site of the RR construct in conventional soybean. Steps (1–5) of PCR-walking. Only EcoRV fragment isolation procedure is shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2656496&req=5

Figure 2: Schematic representation of the PCR-walking approach to isolate the wt allele at the RR locus. Top drawing: original integration site of the RR construct in conventional soybean. Steps (1–5) of PCR-walking. Only EcoRV fragment isolation procedure is shown.
Mentions: Genomic DNA extracted from a conventional soybean genotype was digested with ScaI and EcoRV for which the absence of restriction sites in the 3' genomic flanking sequence had been previously ascertained (Figure 2). DNA fragments were subsequently blunt-ligated with adapters and PCR-walking reactions were performed using forward adapter primers and reverse primers specific for the known sequence flanking the 3' end of the RR insert. PCR products obtained by the nested amplification of the adapter-fragments were purified and characterized, providing sequences for the ScaI/plant DNA and the EcoRV/plant DNA. By combining the two partially overlapping sequences, a 714 bp sequence characteristic of the non-GM genome at the RR insertion site was obtained (Figure 3). Of this, 84 bp corresponded to the flanking region at the 3' end previously reported, whereas the remaining newly described 630 bp represented the wt sequence upstream of the RR insertion site [20]. However, no homology with the plant DNA flanking the 5' end of the insert was found indicating that, following the integration of the transgene, major rearrangements had taken place [20]. In addition, no homology with other sequences in GenBank was detected by BLAST analysis.

Bottom Line: The soybean wt sequence at the RR locus was characterized and found to be highly conserved among conventional genotypes, thus allowing the detection of possibly any soybean non-trait contaminant.In addition, results obtained from the analysis of wt-spiked RR samples demonstrate that it is possible to use the real-time PCR assay to quantify the non-trait contamination with an acceptable degree of accuracy.In principle this approach could be successfully applied to any transgenic event, provided that the wild-type sequence is conserved and single copy.

View Article: PubMed Central - HTML - PubMed

Affiliation: Seed Research and Testing Laboratory (LaRAS), Department of Agroenvironmental Sciences and Technologies (DiSTA), Alma Mater Studiorum University of Bologna, viale Fanin 40, Bologna 40127, Italy. ebattistini@agrsci.unibo.it

ABSTRACT

Background: Trait purity is a key factor for the successful utilization of biotech varieties and is currently assessed by analysis of individual seeds or plants. Here we propose a novel PCR-based approach to test trait purity that can be applied to bulk samples. To this aim the insertion site of a transgene is characterized and the corresponding sequence of the wild-type (wt) allele is used as diagnostic target for amplification. As a demonstration, we developed a real-time quantitative PCR method to test purity of glyphosate tolerant (Roundup Ready, RR) soybean.

Results: The soybean wt sequence at the RR locus was characterized and found to be highly conserved among conventional genotypes, thus allowing the detection of possibly any soybean non-trait contaminant. On the other hand, no amplification product was obtained from RR soybean varieties, indicating that the wt sequence is single copy and represents a suitable marker of conventional soybean presence. In addition, results obtained from the analysis of wt-spiked RR samples demonstrate that it is possible to use the real-time PCR assay to quantify the non-trait contamination with an acceptable degree of accuracy.

Conclusion: In principle this approach could be successfully applied to any transgenic event, provided that the wild-type sequence is conserved and single copy. The main advantages of the assay here described derive from its applicability to bulk samples, which would allow to increase the number of single seeds or plants forming the analytical sample, thus improving accuracy and throughput while containing costs. For these reasons this application of quantitative PCR could represent a useful tool in agricultural biotechnology.

Show MeSH