Simultaneous identification of mutations by dual-parameter multiplex hybridization in peptide nucleic acid-containing...

The physical entrapment of peptide nucleic acids (PNA) in electrophoresis media provides a system for performing real-time hybridization. DNA strands fully complementary to the target PNA are retarded compared to single-nucleotide mismatched strands. A second parameter, that of amplicon length, has been introduced to perform multiplex analyses on several mutations simultaneously. Size fractionation creates a virtual array of PCR products that can hybridize to one of a set of mutation-specific PNAs present within the matrix. Each targeted mutation can be identified by the size of its corresponding amplicon. Its genotype is characterized by its interaction with a specific PNA that gives a visually resolved distinction between wildtype and mutant allele. In contrast to conventional hybridization, heterozygotes are readily distinguished from homozygotes. Using a capillary electrophoresis-based DNA sequencer, this approach has been used to automate the identification of the H63D, S65C, and C282Y mutations in the hereditary hemochromatosis gene.