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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.

Original publication

DOI

10.1002/0471142905.hg0702s14

Type

Journal article

Journal

Curr Protoc Hum Genet

Publication Date

05/2001

Volume

Chapter 7

Pages

Unit - 7.2

Keywords

Base Pair Mismatch, DNA Mutational Analysis, DNA, Complementary, Genetics, Medical, Humans, Molecular Probe Techniques, Phosphorus Radioisotopes, RNA Probes, Ribonuclease, Pancreatic, Ribonucleases