Abstract
Abstract
Background
Copy 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.
Results
Here, 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.
Conclusions
We 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.
Publisher
Springer Science and Business Media LLC
Subject
Plant Science,Genetics,Biotechnology
Reference42 articles.
1. De Buck S, Peck I, De Wilde C, Marjanac G, Nolf J, De Paepe A, Depicker A. Generation of single-copy T-DNA transformants in Arabidopsis by the CRE/loxP recombination-mediated resolution system. Plant Physiol. 2007;145:1171–82.
2. Dai N, Schaffer A, Petreikov M, Shahak Y, Giller Y, Ratner K, Levine A, Granot D. Overexpression of Arabidopsis hexokinase in tomato plants inhibits growth, reduces photosynthesis, and induces rapid senescence. Plant Cell. 1999;11:1253–66.
3. Passricha N, Saifi S, Khatodia S, Tuteja N. Assessing zygosity in progeny of transgenic plants: current methods and perspectives. J Microbiol Methods. 2016;3:e46.
4. Codner GF, Erbs V, Loeffler J, Chessum L, Caulder A, Jullien N, Wells S, Birling M-C, Teboul L. Universal Southern blot protocol with cold or radioactive probes for the validation of alleles obtained by homologous recombination. Methods. 2021;191:59–67.
5. Southern E. Southern blotting. Nat Protoc. 2006;1:518–25.
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