A sequencing method for short RNA using ribonucleases

 The sequence of a short RNA (5-10nt) can be identified by performing an experiment using ribonuclease (RNase) digestion and gel electrophoresis. Some RNases recognize the cleavage site with sequence specific manner; for example, RNase A cleaves a specific RNA sequence that is (C/U)pN. If we use a T7 polymerase and obtain a short RNA such as pppApCpGpGpA that is labeled at the 5' gamma-phosphate (p*ppApCpGpGpA) with radioactive 32P, we use RNase A to estimate the sequence of product. The RNase A digests pppApCpGpGpA into pppApCp. By comparing the mobility of RNase A treated nucleotide to that of non-treated nucleotide in Urea PAGE using 20% polyacrylamide gel containing 7M Urea, we estimate the sequence of the RNA because the mobility of pppApCp is different from that of pppApCpGpGpA. By combining these experiments using several RNases that have different sequence specificity, we can identify the correct sequence of the synthesized short RNA.