Cloning full-length cDNA of grapevine chrome mosaic nepovirus.
Full-length cDNA copies of the genomic RNAs of grapevine chrome mosaic virus were obtained and cloned in Escherichia coli by a one-step procedure. The cloning protocol included size selections by agarose-gel electrophoresis of both the single-stranded and the double-stranded full-length cDNAs. First-strand cDNA synthesis was primed with oligodeoxythymidine while second-strand synthesis was primed with specific synthetic oligodeoxynucleotides, allowing cloning of the 3' poly(A) and of the last 5' nucleotides of the viral RNA template. For the 7.2-kb and 4.4-kb viral RNAs, up to 20% and 80%, respectively, of the clones were found to be full-length. Even for large templates, this procedure allows fast and efficient cloning of full-length cDNAs.
Le Gall
O
O
Station de Pathologie Végétale, INRA, Pont-de-la-Maye, France.
Candresse
T
T
Brault
V
V
Bretout
C
C
Hibrand
L
L
Dunez
J
J
D001483:Base Sequence; D003001:Cloning, Molecular; D004277:DNA, Single-Stranded / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D009029:Mosaic Viruses / Q000235:genetics*; D012367:RNA, Viral / Q000235:genetics
D004277:DNA, Single-Stranded; D004279:DNA, Viral; D012367:RNA, Viral