Two-fold differences are the detection limit for determining transgene copy numbers in plants by real-time PCR

BMC Biotechnology
Ben BubnerIan T Baldwin

Abstract

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. 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. 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 Souther...Continue Reading

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Citations

Sep 14, 2007·Transgenic Research·Takayuki SakuraiTakayuki Shindo
Jul 28, 2010·Proceedings of the National Academy of Sciences of the United States of America·Toshiyuki TsunodaSenji Shirasawa
Sep 15, 2009·The Journal of Biological Chemistry·Christian BachIna Vorberg
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Datasets Mentioned

BETA
AY426758

Methods Mentioned

BETA
PCR
transgenic
transgenics

Software Mentioned

SDS
Primer [UNK]
Primer Express

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