Two-fold Differences Are the Detection Limit for Determining Transgene Copy Numbers in Plants by Real-Time PCR

BMC Biotechnol. 2004 Jul 13;4:14. doi: 10.1186/1472-6750-4-14.


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.

MeSH terms

  • Computer Systems*
  • DNA Probes / genetics
  • DNA, Complementary / genetics
  • DNA, Plant / genetics
  • Gene Amplification / genetics
  • Gene Dosage*
  • Genetic Variation / genetics
  • Genetic Vectors / genetics
  • Heterozygote
  • Plants / genetics*
  • Plants, Genetically Modified
  • Polymerase Chain Reaction / methods*
  • Transgenes / genetics*


  • DNA Probes
  • DNA, Complementary
  • DNA, Plant