The presence of genetically modified organisms (GMO) is commonly
assessed using real-time PCR methods targeting the most common
transgenic elements found in GMOs. Once the presence of GM material
has been established using these screening methods, GMOs are
further identified using a battery of real-time PCR methods, each
being specific of one GM event and usually targeting the junction
of the plant genome and of the transgenic DNA insert. If, using
these specific methods, no GMO could be identified, the presence of
an unauthorized GMO is suspected. In this context, the aim of this
work was to develop a fast and simple method to obtain the sequence
of the transgene and of its junction with plant DNA, with the
presence of a screening sequence as only prior knowledge. An
unauthorized GM petunia, recently found on the French market, was
used as template during the development of this new molecular tool.
The innovative proposed protocol is based on the circularization of
fragmented DNA followed by the amplification of the transgene and
of its flanking regions using long-range inverse PCR. Sequencing
was performed using the Oxford Nanopore MinION technology and a
bioinformatic pipeline was developed.
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