Novel method of detecting single base substitutions in RNA molecules by differential melting behavior in solution.

We have developed a quick and reliable way of detecting point mutations in RNA molecules. This method involves melting RNA-RNA heteroduplexes of varying lengths in a series of tubes containing a stepwise salt or formamide gradient, followed by polyacrylamide gel electrophoresis to distinguish between single- and double-stranded RNA molecules. The manipulations required are technically simple, and the method is sensitive enough to detect destabilization of the highest melting domain of a dsRNA duplex by a single base mismatch. When this method is used in parallel with denaturing gradient gel electrophoresis, which detects point mutations in low-melting domains of duplexes, it should now be possible to rapidly screen for mutations located throughout the length of any RNA molecule whose wild-type sequence is known.