Abstract
In order to strengthen the current genetically modified organism (GMO) detection system for unauthorized GMO, we have recently developed a new workflow based on DNA walking to amplify unknown sequences surrounding a known DNA region. This DNA walking is performed on transgenic elements, commonly found in GMO, that were earlier detected by real-time PCR (qPCR) screening. Previously, we have demonstrated the ability of this approach to detect unauthorized GMO via the identification of unique transgene flanking regions and the unnatural associations of elements from the transgenic cassette. In the present study, we investigate the feasibility to integrate the described workflow with the MinION Next-Generation-Sequencing (NGS). The MinION sequencing platform can provide long read-lengths and deal with heterogenic DNA libraries, allowing for rapid and efficient delivery of sequences of interest. In addition, the ability of this NGS platform to characterize unauthorized and unknown GMO without any a priori knowledge has been assessed.
Highlights
In order to strengthen the current genetically modified organism (GMO) detection system for unauthorized GMO, we have recently developed a new workflow based on DNA walking to amplify unknown sequences surrounding a known DNA region
The present paper is a proof-of-concept study aiming to investigate the potential and benefits of integrating the MinION platform into the strategy described above for the detection and characterization of GMO, including unauthorized ones. This advanced strategy was applied on an European Union (EU) unauthorized genetically modified (GM) Bt rice, allowing to assess its feasibility, and to determine the possibility to prove the presence of GMO through the characterization of their transgene flanking regions and the unnatural associations of transgenic elements from their transgenic cassette, even when no a priori reference information is available
The MinION sequencing run was performed on the PCR products generated from Bt rice using the bidirectional DNA walking methods anchored on the p35S, tNOS and t35S pCAMBIA transgenic elements (Fig. 1A)
Summary
In order to strengthen the current genetically modified organism (GMO) detection system for unauthorized GMO, we have recently developed a new workflow based on DNA walking to amplify unknown sequences surrounding a known DNA region. The present paper is a proof-of-concept study aiming to investigate the potential and benefits of integrating the MinION platform into the strategy described above for the detection and characterization of GMO, including unauthorized ones This advanced strategy was applied on an EU unauthorized genetically modified (GM) Bt rice, allowing to assess its feasibility, and to determine the possibility to prove the presence of GMO through the characterization of their transgene flanking regions and the unnatural associations of transgenic elements from their transgenic cassette, even when no a priori reference information is available
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