Abstract
BackgroundDue to the increased use of genetic modifications in crop improvement, there is a need to develop effective methods for the detection of both known and unknown transgene constructs in plants. We have developed a strategy for detection and characterization of unknown genetic modifications and we present a proof of concept for this method using Arabidopsis thaliana and Oryza sativa (rice). The approach relies on direct hybridization of total genomic DNA to high density microarrays designed to have probes tiled throughout a set of reference sequences.ResultsWe show that by using arrays with 25 basepair probes covering both strands of a set of 235 vectors (2 million basepairs) we can detect transgene sequences in transformed lines of A. thaliana and rice without prior knowledge about the transformation vectors or the T-DNA constructs used to generate the studied plants.ConclusionThe approach should allow the user to detect the presence of transgene sequences and get sufficient information for further characterization of unknown genetic constructs in plants. The only requirements are access to a small amount of pure transgene plant material, that the genetic construct in question is above a certain size (here ≥ 140 basepairs) and that parts of the construct shows some degree of sequence similarity with published genetic elements.
Highlights
Due to the increased use of genetic modifications in crop improvement, there is a need to develop effective methods for the detection of both known and unknown transgene constructs in plants
We show that without prior knowledge about the transgene sequence in question, fragments (≥ 140 bp) of the elements used in the genetic transformation can be detected
Ninety μg seemed to be the upper limit, and this corresponds to roughly 1 fmole haploid A. thaliana genomes and 267 amoles of haploid rice genomes
Summary
Due to the increased use of genetic modifications in crop improvement, there is a need to develop effective methods for the detection of both known and unknown transgene constructs in plants. The diversity of food crop species and traits introduced is rapidly increasing, and with the prospective use of food crop plants in for instance production of pharmaceuticals and biofuels, the risk is increasing for introduction of unauthorized GM material into the foodchain or release of unauthorized GM plants into the environment. Recent examples of such introduction are the presence of event Bt10 in US maize and LL601 rice in US rice exported to Europe [2,3]. With the increased global focus on biosafety, and the realistic scenarios of unintended release of unauthorized and potentially unknown nature of the GM materials, there is a need for availability of technology that rapidly can detect and provide information about a possible unknown GMO
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