A next-generation sequencing method for gene doping detection that distinguishes low levels of plasmid DNA against a background of genomic DNA / Eddy N. de Boer, Petra E. van der Wouden, Lennart F. Johansson, Cleo C. van Diemen, Hidde J. Haisma. - (Gene Therapy (2019) 11 July; p. 1-9).
- DOI: 10.1038/s41434-019-0091-6
Abstract
Gene doping confers health risks for athletes and is a threat to fair competition in sports. Therefore the anti-doping community has given attention on its detection. Previously published polymerase chain reaction-based methodologies for gene doping detection are targeting exon–exon junctions in the intron-less transgene. However, because these junctions are known, it would be relatively easy to evade detection by tampering with the copyDNA sequences. We have developed a targeted nextgeneration sequencing based assay for the detection of all exon–exon junctions of the potential doping genes, EPO, IGF1, IGF2, GH1, and GH2, which is resistant to tampering. Using this assay, all exon–exon junctions of copyDNA of doping genes could be detected with a sensitivity of 1296 copyDNA copies in 1000 ng of genomic DNA. In addition, promotor regions and plasmid-derived sequences are readily detectable in our sequence data. While we show the reliability of our method for a selection of genes, expanding the panel to detect other genes would be straightforward. As we were able to detect plasmidderived sequences, we expect that genes with manipulated junctions, promotor regions, and plasmid or virus-derived sequences will also be readily detected.
