A rapid method for sequencing the 5′- and 3′-termini of double-stranded RNA viral templates using RLM-RACE

Abstract

A protocol is described that permits rapid, inexpensive and reliable synthesis and amplification of 5′- and 3′-terminal cDNAs using viral double-stranded (ds) RNA templates. The method of RNA ligase-mediated amplification of cDNA ends for amplification of single-stranded RNAs (Liu and Gorovsky, 1993) was adapted for dsRNAs to generate the amplicons. These amplicons can be easily cloned and sequenced to complete the sequencing of 5′- and 3′-termini of any particular virus genome. The method is of great utility and can be used on viral genomic dsRNA or viral dsRNA replicative forms when the virion RNA is unavailable.