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 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.
Igloi
G L
GL
Institut für Biologie III, Universität Freiburg, Schänzlestrasse 1, Freiburg, D-79104, Germany. igloi@oligo.biologie.uni-freiburg.de
D001483:Base Sequence; D004247:DNA / Q000737:chemistry / Q000235:genetics; D004252:DNA Mutational Analysis / Q000379:methods*; D019075:Electrophoresis, Capillary; D005838:Genotype; D006432:Hemochromatosis / Q000235:genetics; D006801:Humans; D009154:Mutation; D009693:Nucleic Acid Hybridization / Q000379:methods*; D020135:Peptide Nucleic Acids / Q000235:genetics*; D015203:Reproducibility of Results
D020135:Peptide Nucleic Acids; D004247:DNA