A novel method to map transcripts: evidence for homology between an adenovirus mRNA and discrete multiple regions of the viral genome

Abstract

A method has been devised which permits mapping of transcripts by a two-step hybridization procedure (sandwich hybridization). RNA extracted from cells infected with an adenovirus-SV40 hybrid (Ad2+ND1) was hybridized to restriction endonuclease fragments of adenovirus type 2 (Ad2) DNA immobilized on nitrocellulose filters. RNAs containing both Ad2 and SV40 sequences formed duplexes through their Ad2 sequences, leaving their SV40 sequences as protruding tails. Annealing with 32P-labeled SV40 DNA caused these tails to become labeled, permitting autoradiographic identification of the sequences of Ad2 DNA which are homologous to the RNA. The high sensitivity of this technique, achieved through the use of 32P-labeled RNA of high specific activity, has led to the observation that hybridization of Ad2+ND1 RNA occurs at several locations on the Ad2 genome, in addition to the expected sites of hybridization proximal to the SV40 insertion.