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Construction of Infectious cDNA Clones for RNA Viruses: Turnip Crinkle Virus

 Last Update: 2020-11-14
 Source: Internet
 Author: User

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Reverse genetic approach is widely used in virology as it makes possible direct identification of viral gene function and uses RNA genomes as vectors. Production of infectious c
DNA
clones is an essential step in developing a reverse genetic system for an RNA virus. Here, we present rapid method for generation of infectious
cDNA
clone for Turnip crinkle virus (TCV). The infectious cDNA clone could be used for production of in vitro transcripts with the T7 RNA polymerase which could be used for infection of plants or plant cell protoplasts. The procedure described here includes purification of TCV, viral RNA extraction, reverse transcription,
PCR
amplification of the full-length cDNA copy of TCV linked to a T7 RNA polymerase promoter, cloning into a plasmid vector, in vitro transcription, and selection of infectious clones.

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