Rapid cDNA synthesis and sequencing techniques for the genetic study of bluetongue and other dsRNA viruses.
The genetic study of double-stranded (ds) RNA viruses by sequence analyses of full-length genome segments, or entire viral genomes, has been restricted by the technical difficulties involved in analyses of dsRNA templates. This paper describes improved methods for sequence-independent synthesis of full-length cDNA copies of dsRNA genes and associated sequencing strategies. These methods include an improved version of the 'Single Primer Amplification Technique' (SPAT - [Attoui, H., Billoir, F., Cantaloube, J.F., Biagini, P., de Micco, P. and de Lamballerie, X., 2000. Strategies for the sequence determination of viral dsRNA genomes. J. Virol. Methods 89, 147-158]), which is described here as 'Full-Length Amplification of cDNAs' (FLAC). They also include the development of direct sequencing methods (without cloning) for the resulting full-length cDNAs. These techniques, which are applicable to any viruses with segmented dsRNA genomes and conserved RNA termini, make it possible to generate sequence data rapidly from multiple isolates for molecular epidemiology studies.
Maan
Sushila
S
Department of Arbovirology, Institute for Animal Health, Pirbright Laboratory, Ash Road, Woking, Surrey GU24 0NF, UK.
Rao
Shujing
S
Maan
Narender Singh
NS
Anthony
Simon John
SJ
Attoui
Houssam
H
Samuel
Alan Richard
AR
Mertens
Peter Paul Clement
PP
D001820:Bluetongue virus / Q000235:genetics; D018076:DNA, Complementary / Q000737:chemistry* / Q000235:genetics*; D004587:Electrophoresis, Agar Gel; D004591:Electrophoresis, Polyacrylamide Gel; D016679:Genome, Viral / Q000235:genetics; D012328:RNA Viruses / Q000235:genetics*; D012330:RNA, Double-Stranded / Q000235:genetics*; D012367:RNA, Viral / Q000235:genetics*; D017422:Sequence Analysis, DNA / Q000379:methods*
D018076:DNA, Complementary; D012330:RNA, Double-Stranded; D012367:RNA, Viral