title: Discriminative detection of low-abundance point mutations using a PCR/ligase detection reaction/capillary gel electrophoresis method and fluorescence dual-channel monitoring.
abstract: We applied a facile LIF dual-channel monitoring system recently developed and reported by our group to the polymerase chain reaction/ligase detection reaction/CGE method for detecting low-abundance point mutations present in a wild-type sequence-dominated population. Mutation discrimination limits and signaling fidelity of the analytical system were evaluated using three mutant variations in codon 12 of the K-ras oncogene that have high diagnostic value for colorectal cancer. We demonstrated the high sensitivity of the present method by detecting rare mutations present among an excess of wild-type alleles (one mutation among ~100 normal sequences). This method also simultaneously interrogated the allelic compositions of the test samples with high specificity through spectral discrimination of the dye-tagged ligase detection reaction products using the dual-channel monitoring system.
authors: Department of Chemical Engineering and Materials Science, Faculty of Science and Engineering, Doshisha University, Kyotanabe, Kyoto, Japan.
authors: Mariko
authors: M
authors: Hamada
authors: Koji
authors: K
authors: Shimase
authors: Kazuhiko
authors: K
authors: Tsukagoshi
authors: Masahiko
authors: M
authors: Hashimoto
mesh_terms: D045744:Cell Line, Tumor; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D017931:DNA Primers / Q000235:genetics; D019075:Electrophoresis, Capillary / Q000295:instrumentation*; D004867:Equipment Design; D005453:Fluorescence; D011905:Genes, ras; D006801:Humans; D022521:Ligase Chain Reaction / Q000295:instrumentation*; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000295:instrumentation*
chemical_list: D017931:DNA Primers; D004247:DNA
keywords: Capillary gel electrophoresis; Laser-induced fluorescence; Ligase detection reaction; Point mutation; Single nucleotide variation