Enzymatic RNA sequencing

Publisher Summary The enzymatic RNA sequencing method has the advantage of being available for direct determination of the 5 ' - and 3 ' -terminal nucleotide sequences of RNA. Techniques, such as fingerprint analysis and homochromatographic analysis, have been used for the determination of overlapping. However, these techniques require great skill and much time. For labeling of RNAs with a monophosphate group at their 5′ terminus, this group must first be removed. Dephosphorylation of the 5′-terminal phosphate of RNA molecules is performed in 5/μl of solution, containing 50 mM Tris-HC1, pH 8.0, 0.1 mM EDTA, and 0.2 μg/μl of calf intestinal alkaline phosphatase, by incubation for 30 minutes. The same amount of RNA is digested by incubation with 5 U of RNase PhyM in 10μl of 20 mM sodium citrate, pH 5, containing 7 M urea and 1 mM EDTA. If the conditions for partial digestion of RNA given here are not optimal, the conditions should be estimated by changes of the enzyme-substrate ratio in the reaction mixture.