Strategies for the sequence determination of viral dsRNA genomes

The genetic study of viruses having dsRNA genomes is hampered by the techni cal difficulty of complete sequence determination of dsRNA. Optimised metho ds are described here for sequencing dsRNAs, which meet three different sit uations: (1) genomes that can be obtained in fairly high amounts (> 20 ng p er separated segment); (2) genomes with limited amounts of RNA that can be detected by electrophoretic gel separation and staining; (3) genomes that c annot be detected by electrophoretic gel separation and staining. These met hods include improved Single Primer Amplification Technique protocols, an a daptation of the SMART(TM) methodology, and a new method permitting the sel ective enzymatic removal of dsRNA segments. Strategies permitting adaptatio n of these protocols to the full-length determination of dsRNA viral genome s are described. Each of the protocols is described for sequence determinat ion of a chosen dsRNA virus. (C) 2000 Elsevier Science B.V. All rights rese rved.