Home-made enzymatic premix and Illumina sequencing allow for one-step Gibson assembly and verification of virus infectious clones

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 assemb...

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Veröffentlicht in:Phytopathology research 2020-01, Vol.2 (1), p.1-9, Article 36
Hauptverfasser: Zhao, Mingmin, García, Beatriz, Gallo, Araiz, Tzanetakis, Ioannis E, Simón-Mateo, Carmen, García, Juan Antonio, Pasin, Fabio
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Sprache:eng
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Zusammenfassung: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 -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.
ISSN:2524-4167
2096-5362
2524-4167
DOI:10.1186/s42483-020-00077-4