Challenges for the Characterization of Genetically Modified Animals by the qPCR Technique in the Era of Genomic Editing

Abstract

Characterization of genetically modified organisms through determination of zygosity and transgene integration concerning both copy number and genome site is important for breeding a transgenic line and the use of these organisms in the purpose for which it was obtained. Southern blot, fluorescence in situ hybridization or mating are demanding and time-consuming techniques traditionally used in the characterization of transgenic organisms and, with the exception of mating, give ambiguous results. With the emergence of the real-time quantitative PCR technology, different applications have been described for the analysis of transgenic organisms by determination of several parameters to transgenic analysis. However, the accuracy in quantitation by this method can be influenced in all steps of analysis. This review focuses on the aspects that influence pre-analytical steps (DNA extraction and DNA quantification methods), quantification strategies and data analysis in quantification of copy number and zygosity in transgenic animals.