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 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.
Onderstepoort Veterinary Institute, South Africa. frank@moon.ovi.ac.za
F T
FT
Vreede
M
M
Cloete
G B
GB
Napier
A A
AA
van Dijk
G J
GJ
Viljoen
D000356:African Horse Sickness Virus / Q000235:genetics; D000818:Animals; D015152:Blotting, Northern / Q000662:veterinary; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000032:analysis / Q000096:biosynthesis*; D004587:Electrophoresis, Agar Gel / Q000662:veterinary; D005784:Gene Amplification / Q000235:genetics*; D016679:Genome, Viral; D006736:Horses; D012328:RNA Viruses / Q000235:genetics*
D017931:DNA Primers; D018076:DNA, Complementary