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Using real-time PCR to determine transgene copy number in wheat

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Abstract

Transgene copy number is usually determined by means of Southern blot analysis which can be time consuming and laborious. In this study, quantitative real-time PCR was developed to determine transgene copy number in transgenic wheat. A conserved wheat housekeeping gene,puroindoline-b, was used as an internal control to calculate transgene copy number. Estimated copy number in transgenic lines using real-time quantitative PCR was correlated with actual copy number based on Southern blot analysis. Real-time PCR can analyze hundreds of samples in a day, making it an efficient method for estimating copy number in transgenic wheat.

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Abbreviations

Ct:

threshold cycle

TAMRA:

tetramethylrhodamine

WSMV-CP:

wheat streak mosaic virus coat protein

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Author information

Author notes

  1. Zhiwu Li

    Present address: Plant Sciences Center, Kansas State University, 4024 Throckmorton, 66506, Manhattan, KS, USA

Authors and Affiliations

  1. Plant Pathology Department, Kansas State University, 66502, Manhattan, KS, USA

    Zhiwu Li

  2. The Department of Plant Science, Entomology and Soil, University of Idaho, 83843, Moscow, ID, USA

    Jennifer L. Hansen, Robert S. Zemetra & Philip H. Berger

  3. Statistics Department, Kansas State University, 66502, Manhattan, KS, USA

    Ying Liu

Authors
  1. Zhiwu Li
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  2. Jennifer L. Hansen
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  3. Ying Liu
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  4. Robert S. Zemetra
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  5. Philip H. Berger
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Corresponding author

Correspondence to Zhiwu Li.

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Li, Z., Hansen, J.L., Liu, Y. et al. Using real-time PCR to determine transgene copy number in wheat. Plant Mol Biol Rep 22, 179–188 (2004). https://doi.org/10.1007/BF02772725

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