Detection of mutations by RNase cleavage.
Ribonucleases 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 (32)P-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 Support Protocol 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.
Watkins
H C
HC
University of Oxford, Oxford, UK.
Goldrick
M
M
D020137:Base Pair Mismatch; D004252:DNA Mutational Analysis / Q000379:methods*; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification; D005826:Genetics, Medical; D006801:Humans; D015336:Molecular Probe Techniques; D010761:Phosphorus Radioisotopes; D015347:RNA Probes / Q000235:genetics / Q000302:isolation & purification; D012259:Ribonuclease, Pancreatic; D012260:Ribonucleases
D018076:DNA, Complementary; D010761:Phosphorus Radioisotopes; D015347:RNA Probes; D012260:Ribonucleases; D012259:Ribonuclease, Pancreatic