The Chemical Cleavage of Mismatch for the Detection of Mutations in Long DNA Fragments

Methods to rapidly scan large regions of DNA that are not dependent on highly specific melting temperatures or suitable only for large-scale discovery are important to reduce the amount of sequencing required for DNA samples that appear to contain a mutation. This protocol describes the chemical cleavage of mismatch method to assess if a region of DNA contains a mutation and accurately localize the position of the mutation in the same reaction. To detect mutations, PCR heteroduplexes are incubated with two mismatch-specific reagents. Hydroxylamine modifies mismatched cytosine residues and potassium permanganate modifies mismatched thymine residues. The samples are then incubated with piperidine, which cleaves the DNA backbone at the site of the modified mismatched base. Cleavage products are separated by electrophoresis, revealing the identity and location of the mutation. The chemical cleavage of mismatch method can efficiently detect point mutations as well as insertions and deletions.