An Efficient Cloning Strategy for Viral Double-stranded RNAs with Unknown Sequences.

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  • 未知のウイルス二本鎖RNAゲノムの効率的なクローニング法
  • Efficient Cloning Strategy for Viral Do

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Abstract

A strategy was designed to efficiently clone double-stranded RNAs (dsRNAs) of unknown sequences and very low availability. From rice plants infected with rice black streaked dwarf fijivirus (RBSDV), ten dsRNA genomic segments were extracted directly. The 3' ends of the plus and minus strands of the dsRNAs were polyadenylated and then used as templates for an initial reverse transcription using an oligo-dT-containing adapter primer (AP). The first-strand cDNAs of both polarities were annealed, filled in and amplified by the polymerase chain reaction using one primer containing an adapter region sequence identical to that in the AP. The amplified cDNA products corresponded in size to the full lengths of RBSDV S5, S6, S7, S8, S9 and S10; full length cDNA clones to RBSDV S8, S9 and S10 containing both terminal nucleotide sequences were obtained. Moreover, the nucleotide sequences of six full length cDNA clones were the same as those previously reported. These data indicate that this method may be applicable to full length cDNA cloning of dsRNAs.

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