ArticleBMC biotechnology2004
Two-fold differences are the detection limit for determining transgene copy numbers in plants by real-time PCR.
Article in BMC biotechnology, 2004. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 81 papers.
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81 citing papers in PubMed.
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- Tracing Mobile DNAs: From Molecular to Population Scales.Frontiers in plant science · 2022Review
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- High-Throughput and Accurate Determination of Transgene Copy Number and Zygosity in Transgenic Maize: From DNA Extraction to Data Analysis.International journal of molecular sciences · 2021Article
- microRNA390 modulatesPlant direct · 2021Article
- Specific decorations of 17-hydroxygeranyllinalool diterpene glycosides solve the autotoxicity problem of chemical defense in Nicotiana attenuata.The Plant cell · 2021Article
- Digital PCR (dPCR) and qPCR mediated determination of transgene copy number in the forage legume white clover (Trifolium repens).Molecular biology reports · 2021Article
- Iron incorporation both intra- and extra-cellularly improves the yield and saccharification of switchgrass (Panicum virgatum L.) biomass.Biotechnology for biofuels · 2021Article
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- Association between Oral Pathology, Carotid Stenosis, and Oral Bacterial DNA in Cerebral Thrombi of Patients with Stroke.Stroke research and treatment · 2021Article
- The complete chloroplast genome and phylogenetic analysis ofMitochondrial DNA. Part B, Resources · 2021Article
21 more citing papers are in PubMed but not listed here.
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Abstract
backgroundAfter 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.
resultsWhen 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.
conclusionsDetection 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.
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