Two-fold differences are the detection limit for determining transgene copy numbers in plants by real-time PCR.
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.
Bubner
Ben
B
Department of Molecular Ecology, Max-Planck-Institute for Chemical Ecology, Hans-Knöll-Str, 8, 07745 Jena, Germany. bubner@ice.mpg.de
Gase
Klaus
K
Baldwin
Ian T
IT
D003199:Computer Systems; D015342:DNA Probes / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D018744:DNA, Plant / Q000235:genetics; D005784:Gene Amplification / Q000235:genetics; D018628:Gene Dosage; D014644:Genetic Variation / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D006579:Heterozygote; D010944:Plants / Q000235:genetics*; D030821:Plants, Genetically Modified; D016133:Polymerase Chain Reaction / Q000379:methods*; D019076:Transgenes / Q000235:genetics*
D015342:DNA Probes; D018076:DNA, Complementary; D018744:DNA, Plant