IMPLANT: a new technique for transgene copy number estimation in plants using a single end-point PCR reaction

Plant Methods

Methodology

Hoo Sun Chung¹  Dirk Inzé¹  Charlotte De Bruyn²  Jihae Park³  Stephen Depuydt⁴  Jonas De Saeger⁴  Kai Thoris⁵ 

[1]Department of Plant Biotechnology and Bioinformatics, Ghent University, 9052, Ghent, Belgium

[2]Center for Plant Systems Biology, VIB, 9052, Ghent, Belgium

[3]Laboratory of Plant Growth Analysis, Ghent University Global Campus, 406-840, Incheon, South Korea

[4]Laboratory of Plant Growth Analysis, Ghent University Global Campus, 406-840, Incheon, South Korea

[5]Department of Marine Sciences, Incheon National University, 406-840, Incheon, South Korea

[6]Laboratory of Plant Growth Analysis, Ghent University Global Campus, 406-840, Incheon, South Korea

[7]Department of Plant Biotechnology and Bioinformatics, Ghent University, 9052, Ghent, Belgium

[8]Center for Plant Systems Biology, VIB, 9052, Ghent, Belgium

[9]Laboratory of Plant Growth Analysis, Ghent University Global Campus, 406-840, Incheon, South Korea

[10]Laboratory of Molecular Biology, Wageningen University & Research, 6708 PB, Wageningen, The Netherlands

关键词: Copy number determination;    Competitive PCR;    T-DNA insertions;    IMPLANT;    Genetically modified plants;   

DOI  :  10.1186/s13007-022-00965-0

 received in 2022-01-15, accepted in 2022-11-27,  发布年份 2022

来源: Springer

【 摘 要 】

BackgroundCopy number determination is one of the first steps in the characterization of transgenic plant lines. The classical approach to this, Southern blotting, is time-consuming, expensive and requires massive amounts of high-quality genomic DNA. Other PCR-based techniques are either inaccurate, laborious, or expensive.ResultsHere, we propose a new technique, IMPLANT (Insertion of competitive PCR calibrator for copy number estimation), a competitive PCR-based technique in which the competitor (based on an endogenous gene) is also incorporated in the T-DNA, which then gets integrated in the genome together with the gene of interest. As the number of integrated competitor molecules directly corresponds to the number of transgene copies, the transgene copy number can be determined by a single PCR reaction. We demonstrate that the results of this technique closely correspond with those obtained by segregation analysis in Arabidopsis and digital PCR In rice, indicating that it is a powerful alternative for other techniques for copy number determination.ConclusionsWe show that this technique is not only reliable, but is also faster, easier, and cheaper as compared with other techniques. Accurate results are obtained in both Arabidopsis and rice, but this technique can be easily extended to other organisms and as such can be widely adopted in the field of biotechnology.

【 授权许可】

CC BY   
© The Author(s) 2022

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