title: Chemical cleavage of mismatch (CCM) to locate base mismatches in heteroduplex DNA.
abstract: This protocol describes the use of the chemical cleavage of mismatch (CCM) method to assess whether a region of DNA contains mutations and to localize them. Compared with other mutation-detection techniques (such as single strand-conformation polymorphism (SSCP) analysis, denaturing high-performance liquid chromatography (DHPLC) and denaturing gradient gel electrophoresis (DGGE)) that detect mutations in short DNA fragments and require highly specific melting temperatures, CCM has a higher diagnostic sensitivity suited to the detection of mutations in tumor genes, and can analyze amplicons < or = 2 kb in length. To detect mutations, PCR heteroduplexes are incubated with two mismatch-specific reagents. Hydroxylamine modifies unpaired cytosine and potassium permanganate modifies unpaired thymine. 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 CCM method can efficiently detect point mutations as well as insertions and deletions. This protocol can be completed in 10 h.
authors.affiliation: Genomic Disorders Research Centre, St Vincent's Hospital, PO Box 2900, Victoria 3065, Australia.
authors.forename: Tania
authors.initials: T
authors.lastname: Tabone
authors.forename: Georgina
authors.initials: G
authors.lastname: Sallmann
authors.forename: Maria
authors.initials: M
authors.lastname: Chiotis
authors.forename: Matthew
authors.initials: M
authors.lastname: Law
authors.forename: Richard
authors.initials: R
authors.lastname: Cotton
mesh_terms: D020137:Base Pair Mismatch; D004247:DNA / Q000737:chemistry*; D020180:Heteroduplex Analysis / Q000379:methods*; D019811:Hydroxylamine; D009692:Nucleic Acid Heteroduplexes / Q000737:chemistry*; D011196:Potassium Permanganate
chemical_list: D009692:Nucleic Acid Heteroduplexes; D011196:Potassium Permanganate; D019811:Hydroxylamine; D004247:DNA