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

Bubner, B and Baldwin, I T (2004) Use of real-time PCR for determining copy number and zygosity in transgenic plants. Plant Cell Reports, 23 (5). pp. 263-271.

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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−ΔΔCt ) 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 screening tool for estimating copy numbers of a large number of transformants.

Item Type: Article
Uncontrolled Keywords: Transgenic plants Real-time PCR Copy number Zygosity
Author Affiliation: Max-Planck-Institut für Chemische Ökologie, Hans-Knöll-Strasse 8, 7745, Jena, Germany
Subjects: Crop Improvement
Divisions: General
Depositing User: Mr. SanatKumar Behera
Date Deposited: 16 Jan 2014 08:39
Last Modified: 16 Jan 2014 08:39
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