Abstract
Agrobacterium-mediated transformation has been widely used for genetic engineering of plants and fungi. The use of Agrobacterium as biotechnology workhorse depends heavily on the ability to locate the insertion site of T-DNA. However, it is still challenging to identify T-DNA insertion sites and to link the affected genes to mutant phenotypes. Determination of T-DNA insertion sites requires the PCR-based isolation of flanking regions and the subsequent sequence analysis. Here, we describe TAIL-PCR method for analysis of large-scale samples, interpretation of sequence data, and patterns of T-DNA integration. This information will provide not only technical guidance to experimental trouble-shooting but also practical understanding of procedures to deal with sequence data of T-DNA mutants.
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Acknowledgements
This work was supported by the National Research Foundation of Korea grant funded by the Korea government (2008-0061897, 2013-003196 (to Y.H.L.), and 2013R1A1A1010928 (to J.C)), and the Next-Generation BioGreen 21 Program of Rural Development Administration in Korea (PJ00821201 (to Y.H.L)).
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Choi, J., Jeon, J., Lee, YH. (2015). Identification of T-DNA Integration Sites: TAIL-PCR and Sequence Analysis. In: van den Berg, M., Maruthachalam, K. (eds) Genetic Transformation Systems in Fungi, Volume 2. Fungal Biology. Springer, Cham. https://doi.org/10.1007/978-3-319-10503-1_19
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DOI: https://doi.org/10.1007/978-3-319-10503-1_19
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