A method for determining zygosity of transgenic zebrafish by TaqMan real-time PCR.

When producing a genetically modified organism, intended genes are often integrated into a target genome by random insertions. Subsequently, it is often desirable to know the gene copy number of the transgenic organism and the zygosity of its offspring. Because of the random insertions, the estimation can be made only by quantitative measurement of the genes. Even though TaqMan real-time PCR has been used in gene expression analysis, it is routinely used to quantify differences larger than twofold or more than one PCR cycle. In this study, we employed TaqMan quantitative PCR to determine zygosity of transgenic fluorescent zebrafish in which a homozygote and a hemizygote differ by only twofold. We measured relative quantities of the transgene by taking the threshold cycle (Ct) of both the transgene and an internal control zebrafish genomic DNA. Using scatterplots and statistical inference, we demonstrated that homozygotes and hemizygotes could be differentiated unambiguously when multiple measurements were taken. We discuss the relationship between the repetitive measurements and TaqMan precision with a statistical model. The result illustrates that the method can be extended to some areas that require even higher precision such as determining the polyploidy of an organism.