Abstract
Engineered plants have been widely produced for fundamental and practical use. Several methods have been developed for genetically modified crop detection and quantification; however; they still laborious and expensive. Efforts are needed to set-up diagnosis-oriented techniques as alternatives to overcome DNA extraction which remains a tedious and time-consuming procedure. Here, we established a standard direct PCR workflow using a regular Taq polymerase without prior DNA purification over a wide range of plant species. Only a small amount of fresh tissue allowed direct amplification of target gene sequences. Evaluation of accuracy, sensitivity, and reproducibility of direct PCR assay was investigated for proof-of-concept, and subsequently applied to gene detection assays and rapid transgenic revealing. The newly established method achieved full success and has amplified constitutive housekeeping genes from several plant specimens in a reproducible manner with high-quality sequencing profiles. In our case, the screening of transgenic plants confirmed that both the gfp-ER reporter gene and the npt II selectable marker were integrated into the plant genome. This direct PCR approach provides a powerful tool for large-scale PCR-based gene detection making DNA purification irrelevant. It could be easily implemented for downstream applications in the field of genetic fingerprinting, plant biotechnology, and functional genomics.
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Acknowledgements
We are grateful to Dr. Andrea Devlin for English editing and scientific proof reading this manuscript. We would like to thank Dr. Michael Florian Mette from Leibniz Institute of Plant Genetics and Crop Plant Research (IPK-Gatersleben, Germany) for helpful suggestions.
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This work was supported by the Tunisian Ministry of Higher Education and Scientific Research.
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All authors contributed to the study conception and design. Material preparation, experiments, data collection, analysis and manuscript drafting were performed by ABA. ABA and AM discussed the results, read and approved the final manuscript.
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Ben-Amar, A., Mliki, A. Timely gene detection assay and reliable screening of genetically engineered plants using an improved direct PCR-based technology. Transgenic Res 30, 263–274 (2021). https://doi.org/10.1007/s11248-021-00250-1
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DOI: https://doi.org/10.1007/s11248-021-00250-1