Primer Extension Assay

The indicated 5'-end-labeled double stranded primer［32 P］DNA was denatured；the strands were separated and hybridized to R.sphaeroides RNA in the same manner as for the S-1 protection assay.The primer extension assay was performed using the procedure of Mullet et al.［16］.The reaction proceeded in 20 μl of 50 mmol/L Tris-HCl，pH 8.3，70 mmol/L KCl，5 mmol/L MgCl2，10 mmol/L dithiothreitol，0.1 mg/ml bovine serum albumin，1 mmol/L of each dNTP，and 20 units of avian myeloblastosis virus reverse transcriptase at 37℃for 60 min.After phenol extraction and ethanol precipitation，the nucleic acids were analyzed on polyacrylamide gels by the same procedure used for the S-l protection analysis.(https://www.daowen.com)