Abstract
The production of geminiviral infectious clones provides a standardized inoculum for use in several host–virus studies. Geminiviruses present either one (monopartite) or two (bipartite) circular single-stranded DNA components, which commonly range from 2.6 to 2.8 kb. Cloning of a monomeric genome is useful for obtaining its precise sequence. For infectious clones, however, it is essential that more than one copy of the genome, more specifically of the origin of replication, is present in order to guarantee the production of full-length progeny DNA. Here, the complete process of preparing infectious geminiviral clones is described starting from the DNA extraction and selection of restriction endonucleases followed by two protocols for constructing dimeric clones: restriction endonuclease digestion and ligation (1) and Gibson Assembly (2).
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Acknowledgments
We thank all the people in the laboratory, from the past to the present, who engaged in the task of producing infectious clones of geminiviruses. We also thank Dorian Yest Melo Silva for the gel picture in Fig. 2. This work was funded by FAP-DF grants 00193-00000229/2021-21, 0193.000872/2021-54 and 0193.001023/2021-18, and all other grants related to projects on geminivirus diseases. The first author has a scholarship from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). AKI-N is a CNPq fellow.
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Morais, I.J., Inoue-Nagata, A.K., Nakasu, E.Y.T. (2024). Construction of Geminivirus Infectious Clones for Agroinoculation into Plants. In: Fontes, E.P., Mäkinen, K. (eds) Plant-Virus Interactions. Methods in Molecular Biology, vol 2724. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3485-1_4
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DOI: https://doi.org/10.1007/978-1-0716-3485-1_4
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