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Título: | Home-made enzymatic premix and Illumina sequencing allow for one-step Gibson assembly and verification of virus infectious clones |
Autor: | Zhao, Mingmin; García, Beatriz; Gallo, Araiz; Tzanetakis, Ioannis E.; Simón-Mateo, Carmen; García, Juan Antonio CSIC ORCID ; Pasin, Fabio CSIC ORCID | Palabras clave: | Virus infectious clone Agro-infection Gibson assembly Illumina sequencing Home-made cloning reagents Potyvirus Tobacco vein mottling virus |
Fecha de publicación: | 2020 | Editor: | Springer Nature | Citación: | Phytopathology Research 2: 36 (2020) | Resumen: | An unprecedented number of viruses have been discovered by leveraging advances in high-throughput sequencing. Infectious clone technology is a universal approach that facilitates the study of biology and role in disease of viruses. In recent years homology-based cloning methods such as Gibson assembly have been used to generate virus infectious clones. We detail herein the preparation of home-made cloning materials for Gibson assembly. The home-made materials were used in one-step generation of the infectious cDNA clone of a plant RNA virus into a T-DNA binary vector. The clone was verified by a single Illumina reaction and a de novo read assembly approach that required no primer walking, custom primers or reference sequences. Clone infectivity was finally confirmed by Agrobacterium-mediated delivery to host plants. We anticipate that the convenient home-made materials, one-step cloning and Illumina verification strategies described herein will accelerate characterization of viruses and their role in disease development. | Descripción: | © The Author(s). | Versión del editor: | http://dx.doi.org/10.1186/s42483-020-00077-4 | URI: | http://hdl.handle.net/10261/239422 | DOI: | 10.1186/s42483-020-00077-4 | Identificadores: | doi: 10.1186/s42483-020-00077-4 issn: 2524-4167 |
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