Abstract
The EU directive 2001/18/EC requires any genetically modified (GM) event to be stable. In the present work, a targeted Next-Generation Sequencing (NGS) approach using barcodes to specifically tag each individual DNA molecules during library preparation was implemented to detect mutations taking into account the background noise due to amplification and sequencing errors. The method was first showed to be efficient in detecting the mutations in synthetic samples prepared with custom-synthesized mutated or non-mutated P35S sequences mixed in different proportions. The genetic stability of a portion of the P35S promoter targeted for GM detection was then analyzed in GM flour samples. Several low frequency mutations were detected in the P35S sequences. Some mutated nucleotides were located within the primers and probes used in the P35S diagnostic test. If present not as somatic mutations but as the consensus sequence of some individuals, these mutations could influence the efficiency of the P35S real time PCR diagnostic test. This methodology could be implemented in genetic stability studies of GM inserts but also to detect single nucleotide mutant GM plants produced using “new breeding techniques”.
Highlights
Since the first year of commercialization in 1996, the development of genetically modified (GM) crops has constantly grown in terms of cultivated land area, plant species and diversity of GM characteristics
High-Resolution Melting (HRM) being based on the melting properties of the double stranded DNA molecules synthetized during PCR amplification, the obtained result is characteristic of the consensus sequence of the DNA molecules present in the sample and does not provide information on the variability of the DNA sequences within the sample
The application of Next Generation Sequencing (NGS) approaches on DNA extracts from flour, which are obtained from populations of seeds from various plants and environments, constitutes a method of choice to characterize the stability of a GM insert
Summary
Since the first year of commercialization in 1996, the development of genetically modified (GM) crops has constantly grown in terms of cultivated land area, plant species and diversity of GM characteristics. Real-time PCR coupled with HRM is a rapid, simple, robust and sensitive method for detecting mutation and enabling the screening of many samples[13] This method does not provide information about nucleotide sequences and requires subsequent sequencing. HRM being based on the melting properties of the double stranded DNA molecules synthetized during PCR amplification, the obtained result is characteristic of the consensus sequence of the DNA molecules present in the sample and does not provide information on the variability of the DNA sequences within the sample It is only suitable for the analysis of separate individuals (seeds or plants). Stalhberg et al (2016, 2017) developed a barcode-based approach called “Simple, Multiplexed, PCR-based barcoding of DNA for Sensitive mutation detection using Sequencing” (SiMSen-Seq) Their method uses reduced primer concentration, elongated PCR extension times and hairpin-protected barcode primers to enable simple and short library construction protocol[20]. The variability of a portion of the P35S promoter targeted for GM detection was analyzed in GM flour samples
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