Original reverse transcription polymerase chain reaction method to obtain the full-length cDNA of rice tungro spherical virus.
A two-step reverse transcription reaction combined with long PCR was developed in order to obtain the full-length cDNA from the 12.2 kbp genomic RNA of rice tungro spherical virus. A first step reverse transcription, performed at 45 degrees C using a reverse transcriptase deprived of RNase H activity, allowed the synthesis of a nearly full-length cDNA of 11.7 kbp. A second step reaction, carried out at 65 degrees C using a thermostable polymerase, was necessary to destabilise secondary structures present at the 5' extremity of the RNA template which hampered the reverse transcription reaction in this region. The full-length cDNA obtained by the two-step reverse transcription was amplified successfully by long PCR and subsequently cloned into a plasmid vector. The cloned cDNA showed toxicity and proved to be unstable when amplified in E. coli.
Perrin
Y
Y
Department of Virus Research, John Innes Centre, Norwich Research Park, UK. yolande.perrin@bbsrc.ac.uk
Hull
R
R
D003001:Cloning, Molecular; D018076:DNA, Complementary; D004279:DNA, Viral / Q000138:chemical synthesis*; D005822:Genetic Vectors; D012275:Oryza / Q000821:virology*; D010957:Plasmids; D012367:RNA, Viral; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D019192:Sequivirus / Q000235:genetics*
D018076:DNA, Complementary; D004279:DNA, Viral; D012367:RNA, Viral