Construction of infectious clones for RNA viruses: TMV.
The generation of infectious clones is routinely the first step for reverse genetic studies of RNA plant virus gene and sequence function. The procedure given here, details the creation of cDNA clones of tobacco mosaic virus, from which infectious transcripts can be generated in vitro with T7 RNA polymerase. The procedure describes methods for virion purification, viral RNA extraction, reverse transcription, PCR amplification of genomic cDNA fragments, generation of a full-length cDNA clone under the control of a T7 promoter, in vitro transcription, and infectivity testing.
Chapman
Sean N
SN
Plant Pathology Programme, Scottish Crop Research Institute, Scottish Crop Research Institute, Invergowrie, Dundee, DD2 5DA, UK.
D003001:Cloning, Molecular / Q000379:methods; D017931:DNA Primers; D018076:DNA, Complementary / Q000235:genetics; D012321:DNA-Directed RNA Polymerases / Q000235:genetics; D005784:Gene Amplification; D010935:Plant Diseases / Q000821:virology; D011401:Promoter Regions, Genetic; D012328:RNA Viruses / Q000235:genetics / Q000472:pathogenicity; D012367:RNA, Viral / Q000235:genetics / Q000302:isolation & purification; D012194:RNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism; D014026:Nicotiana / Q000821:virology; D014027:Tobacco Mosaic Virus / Q000235:genetics* / Q000472:pathogenicity*; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000235:genetics
D017931:DNA Primers; D018076:DNA, Complementary; D012367:RNA, Viral; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012194:RNA-Directed DNA Polymerase; D012321:DNA-Directed RNA Polymerases