RNase A Cleavage Method. Mispaired nucleotides within heteroduplexes are modified by chemical agents by using Maxam-Gilbert sequencing chemistry. Hydroxylamine reacts with mispaired cytosine residues, osmium tetroxide with mispaired thymidine residues. Radioactively labeled fragments are used. Silver staining or fluorescence labeling are also applied.
All possible mutations are detectable, when both sense and antisense strands are analyzed.
Heteroduplexes generated by heat denaturation of PCR products of polymorphic DNA or wild-type and mutant alleles, respectively, are incubated and cleaved by either the bacteriophage T4 endonuclease YII or T7 endonuclease I. DNA fragments are analyzed by electrophoresis. Silver staining should be feasible. Mutations are detectable in PCR products between 88 and 940 bp or up to 1.5 kb. CFLP patterns are resolved on short , denaturing PAGE . Detection of fragments proceeds through labeling of one of the PCR primers.