Molecular cloning of a human rotavirus genome

The genes of a field isolate of human rotavirus were cloned into pBR322. The strategy used involved polyadenylation of denatured double-stranded (ds) genomic RNA, followed by cDNA synthesis using reverse transcriptase. Oligo(dC) tails were added to the 3' end of the single-stranded cDNA and then separated by alkaline agarose electrophoresis. Sized cDNA of both polarities were allowed to hybridize and inserted into the PstI site of pBR322. Transformations done with sized cDNA always resulted in the selection of hybrid plasmids carrying inserts with a size representative of the original cDNA. Four individual clones were selected for preliminary characterization. One clone has an insert of 1360 base pairs (bp) and corresponds to gene 6. The second clone has an insert of 1140 bp and corresponds to one of the genes in the triplet 7-8-9. The other two genes, with inserts of 780 and 660 bp, were identified as corresponding to dsRNA segments 10 and 11.