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,Hidde J Haisma,Lennart F Johansson,Cleo C Van Diemen,Petra E Van Der Wouden

doi:10.1038/s41434-019-0091-6

Eddy N De Boer, Hidde J Haisma + Show 3 more

Open Access

https://doi.org/10.1038/s41434-019-0091-6

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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 next-generation 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 plasmid-derived sequences, we expect that genes with manipulated junctions, promotor regions, and plasmid or virus-derived sequences will also be readily detected.

Highlights

Doping is a threat to the integrity of sport and the health of athletes
Plasmids of the potential doping genes EPO, growth hormone 1 (GH1), growth hormone 2 (GH2), IGF1, and IGF2 were mixed with high-molecular genomic DNA (gDNA) from a pool of donors in a percentage ranging from 0.01 to 1 for this proof-of-principle study
Gene doping-derived proteins produced by the body of the athlete would, in most cases, be indistinguishable from endogenous proteins, and detection of gene doping should take place at the DNA level [5,6,7, 10]

Summary

Introduction

Doping is a threat to the integrity of sport and the health of athletes. There is no current evidence that gene doping has ever been used, continuous improvements in gene-therapy techniques increase the likelihood of abuse. Since 2004, the anti-doping community has been given attention on developing a test for the detection of gene doping [1, 2]. These authors contributed : Cleo C. van Diemen, Hidde J.

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