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

The physical entrapment of peptide nucleic acids (PNA) in electrophoresis m edia provides a system for performing real-time hybridization. DNA strands fully complementary to the target PNA are retarded compared to single-nucle otide mismatched strands. A second parameter, that of amplicon length, has been introduced to perform multiplex analyses on several mutations simultan eously. Size fractionation creates a virtual array of PCR products that can hybridize to one of a set of mutation-specific PNAs present within the mat ric. Each targeted mutation can be identified by the size of its correspond ing amplicon. Its genotype is characterized by its interaction with a speci fic PNA that gives a visually resolved distinction between wildtype and mut ant allele. In: contrast to conventional hybridization, heterozygotes are r eadily distinguished from homozygotes. Using a capillary electrophoresis-ba sed DNA sequencer, this approach has been used to automate the identificati on of the H63D, S65C, and C282Y mutations in the hereditary hemochromatosis gene. (C) 2001 Academic Press.