Journal
JOURNAL OF VIROLOGICAL METHODS
Volume 143, Issue 2, Pages 132-139Publisher
ELSEVIER SCIENCE BV
DOI: 10.1016/j.jviromet.2007.02.016
Keywords
bluetongue virus; reovirus; dsRNA virus; cDNA; rapid sequencing; FLAC
Funding
- Biotechnology and Biological Sciences Research Council [BBS/E/I/00000999] Funding Source: researchfish
- Biotechnology and Biological Sciences Research Council [BBS/E/I/00000999] Funding Source: Medline
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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. (C) 2007 Elsevier B.V. All rights reserved.
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