33. Construction of Infectious cDNA Clones for RNA Viruses: Turnip Crinkle Virus
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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 cDNA 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.
Series: Methods in Molecular Biology | Volume: 451 | Pub. Date: Apr-01-2008 | Page Range: 491-502 | DOI: 10.1007/978-1-59745-102-4_33
Subject: Plant Sciences
Key Words: Plant RNA virus - Turnip crinkle virus - Carmovirus - Infectious cDNA clone - Genomic RNA - Reverse transcription - Polymerase chain reaction - T7 RNA polymerase - In vitro transcription - Reverse genetic system
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