Tail sequencing. (A) Protocol. Luciferase reporter mRNA was injected and RNAs prepared after a 16-h incubation. Step (1): RNAs were ligated to the 3′-amino modified DNA primer P1. Step (2): Reverse transcriptase was used to synthesize DNAs complementary to the substrate RNAs. Oligonucloeotide P1′, which is complementary to primer P1, was used as a primer for this step. Step (3): cDNAs were amplified by PCR reactions using a substrate specific primer, P2, and P1′. PCR products contain tails of the ligated products. Step (4): PCR reaction products were cloned into vectors using blunt-end ligation and transformed in E. coli. DNA was prepared from independent E. coli transformants, and the sequence of the insert in the plasmid determined. (B) Results. The tethered enzymes add poly(U). Sequences obtained from the RNAs products of reactions using eight different tethered enzymes, as indicated in the figures. The sequences in the figure correspond to that present in the RNA. The number of clones obtained is given in the second column: thus, with At1, seven clones contained 13 T residues.
