Microarray-based method for detection of unknown genetic modifications

Abstract

Background: 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 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.

Results: We 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.

Conclusion: The 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.

Figure 1

Sequences detected by the arrays. The detected sequence windows were ranked according to the number of probes that defined them and the four highest ranked windows with non-overlapping hits to the T-DNA sequences are shown (for a complete list of positive windows, [see Additional file 2]. The scale for the detected windows is 4× the scale for the T-DNA constructs. Hatch marks correspond to point mutations. The bar diagram showing signal/noise ratio for matching probes (averaged across sets of 10 probes) has been included for the ida dataset as an example. The 1,239 bp vector backbone fragment in ida has been described previously [28]. NOS – nopaline synthase. oct – octopine synthase. nptII – neomycin phosphotransferase II. CaMV 35S – cauliflower mosaic virus 35S promoter. uidA – beta glucuronidase. CaMV polyA – cauliflower mosaic virus 3' UTR polyadenylation signal. hptII – hygromycin phosphotransferase. CaMV 35S (double enhancer) – cauliflower mosaic virus 35S promoter, double enhancer version. Ubi – maize ubiquitin. CecA – cecropin A.