Detection of Mutations by RNase Cleavage

AbstractRibonucleases can specifically recognize and cleave RNA at the site of sequence mismatches in RNA‐DNA or RNA‐RNA hybrids. The cleavage products are then characterized by gel electrophoresis. In this unit, a procedure is presented for RNase cleavage of 32P‐labeled riboprobes (transcribed from a cloned copy of the normal sequence) that have been annealed to amplified sequences of a candidate gene or cDNA obtained from affected individuals. A explains how to prepare riboprobes from a genomic or cDNA template obtained from a nonmutant individual. An alternate protocol describes cleavage of RNARNA hybrids using a nonisotopic RNase cleavage mutation assay. Sequential PCR and in vitro transcription steps generate sufficient quantities of duplex RNA targets so that the cleavage products can be detected on a gel by ethidium bromide staining. The unit also discusses the use of alternative ribonucleases for cleaving singlebase mismatches.