Cloning full-length cDNA of grapevine chrome mosaic nepovirus

Gene. 1988 Dec 15;73(1):67-75. doi: 10.1016/0378-1119(88)90313-7.

Abstract

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.

MeSH terms

  • Base Sequence
  • Cloning, Molecular*
  • DNA, Single-Stranded / genetics
  • DNA, Viral / genetics*
  • DNA, Viral / isolation & purification
  • Molecular Sequence Data
  • Mosaic Viruses / genetics*
  • RNA, Viral / genetics

Substances

  • DNA, Single-Stranded
  • DNA, Viral
  • RNA, Viral