Rapid cDNA synthesis and sequencing techniques for the genetic study of bluetongue and other dsRNA viruses.
J Virol Methods
; 143(2): 132-9, 2007 Aug.
Article
in En
| MEDLINE
| ID: mdl-17433453
ABSTRACT
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.
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Collection:
01-internacional
Database:
MEDLINE
Main subject:
RNA Viruses
/
RNA, Double-Stranded
/
RNA, Viral
/
Sequence Analysis, DNA
/
DNA, Complementary
Language:
En
Journal:
J Virol Methods
Year:
2007
Document type:
Article
Affiliation country: