Virus Genes

, Volume 1, Issue 3, pp 243–253 | Cite as

Construction of full-length cDNA copies of viral double-stranded RNA

  • Martin E. Nemeroff
  • Dennis F. Pietras
  • Jeremy A. Bruenn
Article

Abstract

A method is described for the construction of full-length cDNA clones of dsRNAs. All dsRNA viruses have a capsid-associated transcriptase that is responsible for synthesis of the plus strand that is then extruded from viral particles. We have used in vitro transcripts synthesized by the segmentedSaccharomyces cerevisiae virus (ScV) as templates for first-strand cDNA synthesis. Synthesis was primed by a 33-base synthetic oligonucleotide. This contained 27 nucleotides complementary to the 3′ end of the plus strand from one ScV viral dsRNA segment (S14), and 6 additional nucleotides encoding anXbaI restriction site at the 5′ end. The second cDNA strand was synthesized using a similarXbaI linker-synthetic oligonucleotide and the ds cDNA was cloned by standard ligation techniques. All four cDNA plasmid isolates characterized by sequence analysis contained the complete 5′ end sequence of S14. Two of these were complete at the 3′ end, and one lacked a single base here. Of these four clones, one also retained theXbaI sites at either end. Preparing full-length cDNA clones with unique restriction-site linkers by the use of synthetic oligonucletides allows for easier screening for complete cDNA clones if neither the vector nor the cDNA has the chosen restriction site. It also provides for easier sequence analysis and manipulation of the genome for later studies, such as cloning into expression vectors. This method is more efficient than any previously described for production of full-sized cDNA clones.

Key words

cDNA cloning yeast virus dsRNA in vitro transcripts oligonucleotide primers reverse transcriptase 

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Copyright information

© Martinus Nijhoff Publishing 1988

Authors and Affiliations

  • Martin E. Nemeroff
    • 1
  • Dennis F. Pietras
    • 1
  • Jeremy A. Bruenn
    • 1
  1. 1.Department of Biological SciencesState University of New York at BuffaloBuffaloUSA

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