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

ABSTRACT: 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

SUBMITTER: Zhao M 

PROVIDER: S-EPMC7990137 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature