Abstract
The dideoxynucleotide method for sequencing DNA developed by Sanger et al. was modified to allow sequence analysis of poliovirus RNA without recourse to cloning. The method involves reverse transcription of poliovirus RNA followed by c[complementary]DNA-dependent DNA synthesis in the presence of unlabeled dNTP [deoxynucleoside triphosphates] and 2'',3''-dideoxynucleoside triphosphates, with Escherichia coli DNA polymerase I (Klenow) used to catalyze the reaction. DNA synthesis is primed by 5''-32P-labeled RNase T1- or RNase A-resistant oligonucleotides generated from poliovirus RNA. The sequence of 1060 nucleotides preceding the 3''-terminal poly(A) is presented. Based on the position of termination codons, viral translation terminates at nucleotide -562.