SEQUENCE-INDEPENDENT AMPLIFICATION AND CLONING OF LARGE DSRNA VIRUS GENOME SEGMENTS BY POLY(DA)-OLIGONUCLEOTIDE LIGATION

A strategy was developed for sequence-independent synthesis and amplif ication of full-length cDNA of 3-4 kb genes of dsRNA viruses. The meth od of single primer amplification (Lambden et al., 1992) was adapted b y the inclusion of a 3' poly(A) tail to an oligonucleotide ligated to dsRNA genome segments as a template for oligo(dT)-primed cDNA synthesi s. Full-length copies of the largest genome segments, 1 (4 kb) and 2 ( 3 kb), of African horst sickness virus (AHSV) have been cloned, termin ally sequenced and expressed in vitro. (C) 1998 Elsevier Science B.V. All rights reserved.