Use of real-time PCR for determining copy number and zygosity in transgenic plants

Plant Cell Reports
Ben Bubner, Ian T Baldwin

Abstract

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 screeni...Continue Reading

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Citations

Nov 11, 2010·Fish Physiology and Biochemistry·Guofang ZhongHongqi Zhou
Sep 11, 2012·Plant Molecular Biology·Shareena SreedharanThumballi R Ganapathi
Jul 7, 2011·Bulletin of Entomological Research·O MittapalliM A R Mian
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Jan 13, 2006·BMC Genetics·Agnes SzilagyiZsolt Ronai
Mar 10, 2009·BMC Biotechnology·Elena Battistini, Enrico Noli
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Jan 24, 2009·Clinical Biochemistry·Maria TimofeevaAngela Risch
Dec 22, 2005·The Plant Journal : for Cell and Molecular Biology·Anja PascholdIan T Baldwin

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