Sequence-independent amplification and cloning of large dsRNA virus genome segments by poly(dA)-oligonucleotide ligation

Abstract

A strategy was developed for sequence-independent synthesis and amplification of full-length cDNA of 3–4 kb genes of dsRNA viruses. The method of single primer amplification ( Lambden et al., 1992) was adapted by the inclusion of a 3′ poly(A) tail to an oligonucleotide ligated to dsRNA genome segments as a template for oligo(dT)-primed cDNA synthesis. Full-length copies of the largest genome segments, 1 (4 kb) and 2 (3 kb), of African horse sickness virus (AHSV) have been cloned, terminally sequenced and expressed in vitro.