Author: Zhao, Mingmin; GarcÃa, Beatriz; Gallo, Araiz; Tzanetakis, Ioannis E.; Simón-Mateo, Carmen; GarcÃa, Juan Antonio; Pasin, Fabio
Title: Home-made enzymatic premix and Illumina sequencing allow for one-step Gibson assembly and verification of virus infectious clones Cord-id: y1qxmwwt Document date: 2020_12_3
ID: y1qxmwwt
Snippet: 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
Document: 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.
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