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This review examines how real-time PCR can be used to determine copy number and zygosity in transgenic plants. Distinguishing between plants that harbor one and two copies of a transgene or are hemizygous and homozygous requires the ability to routinely distinguish twofold differences, a detection difference which approaches the resolution of PCR-based quantification methods. After explaining the basic principles, especially the threshold cycle (Ct value) as the basic measuring unit in real-time PCR, we introduce three quantitation methods currently in use. While the absolute and relative standard curve approaches are qualitative methods that distinguish high-copy from low-copy transformants, the comparative (2(-DeltaDeltaCt)) method with double-dye oligonucleotides (TaqMan probes) is able to detect twofold differences. In order to obtain reliable results, Ct values for an amplicon should be below 25 and the standard deviation below 0.3. Although real-time PCR can deliver exact copy number determinations, the procedure is not fail-safe. Therefore, real-time PCR should to be viewed as complementary to--rather than as a replacement of--other methods such as Southern analysis, but it is particularly useful as a preliminary screening tool for estimating copy numbers of a large number of transformants.

Background: After transformation, plants that are homozygous and contain one copy of the transgene are typically selected for further study. If real-time PCR is to be used to determine copy number and zygosity, it must be able to distinguish hemizygous from homozygous and one-copy from two-copy plants. That is, it must be able to detect two-fold differences., Results: When transgenic Nicotiana attenuata plants which had been previously determined by Southern analysis to contain one or two copies of the transgene, were analyzed by real-time PCR (2-delta delta Ct method), the method failed to confirm the results from the Southern analysis. In a second data set we analyzed offspring of a hemizygous one-copy plant, which were expected to segregate into three groups of offspring in a 1:2:1 ratio: no transgene, hemizygous, homozygous. Because it was not possible to distinguish homozygous from hemizygous plants with real-time PCR, we could not verify this segregation ratio., Conclusions: Detection of two-fold differences by real-time PCR is essential if this procedure is to be used for the characterization of transgenic plants. However, given the high variability between replicates, a detection of two-fold differences is in many cases not possible; in such cases Southern analysis is the more reliable procedure.