Fig. 1
From: Multiplex detection of seven transgenes for human gene doping analysis
Illustration of the gene doping (GD) panel detection strategy (a) and process chart of the method (b). a) An artificial transgene can be distinguished from an endogenous gene due to the presence of intron-lacking exon-exon junctions (EEJ, exons are displayed as grey bars). The GD panel approach applies a high multiplex PCR-amplification step followed by a single base extension (SBE)-reaction over EEJ-sites. The PCR amplicons are indicated as blue and yellow bars, primers as blue and yellow small horizontal hooks. An exemplary SBE with an “A” or “C” nucleotide of an EEJ-sequence-binding extension primer is indicated with grey arrows. (b) Upon DNA-extraction via a spin-column based procedure, the multiplex PCR is run. The sensitivity of the method is increased by addition of a second PCR step into the method. Upon dephosphorylation and thus inactivation of excessive dNTPs via shrimp alkaline phosphatase (SAP), the SBE-reaction (EXT) is performed via addition of the extension primers and ddNTPs. The SBE-products can then be detected via MALDI-TOF MS as extended primers will lead to a mass shift in the MS-detection spectra. Approximate run times of the different steps are indicated in hours (h).
