title: Development of a ligase detection reaction/CGE method using a LIF dual-channel detection system for direct identification of allelic composition of mutated DNA in a mixed population of excess wild-type DNA.
abstract: We developed an inexpensive LIF dual-channel detection system and applied it to a ligase detection reaction (LDR)/CGE method to identify the allelic composition of low-abundance point mutations in a large excess of wild-type DNA in a single reaction with a high degree of certainty. Ligation was performed in a tube with a nonlabeled common primer and multiplex discriminating primers, each labeled with a different standard fluorophore. The discriminating primers were directed against three mutant variations in codon 12 of the K-ras oncogene that have a high diagnostic value for colorectal cancer. LDR products generated from a particular K-ras mutation through successful ligation events were separated from remaining discriminating primers by CGE, followed by LIF detection using the new system, which consists of two photomultiplier tubes, each with a unique optical filter. Each fluorophore label conjugated to the corresponding LDR product produced a distinct fluorescence signal intensity ratio from the two detection channels, allowing spectral discrimination of the three labels. The ability of this system to detect point mutations in a wild-type sequence-dominated population, and to disclose their allelic composition, was thus demonstrated successfully.
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: Keiichi
authors: K
authors: Noda
authors: Kazuhiko
authors: K
authors: Tsukagoshi
authors: Masahiko
authors: M
authors: Hashimoto
mesh_terms: D000483:Alleles; D004247:DNA / Q000235:genetics* / Q000378:metabolism; D011088:DNA Ligases / Q000378:metabolism; D019075:Electrophoresis, Capillary / Q000295:instrumentation* / Q000379:methods; D004867:Equipment Design; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000032:analysis; D011905:Genes, ras; D007834:Lasers; D017354:Point Mutation
chemical_list: D005456:Fluorescent Dyes; D004247:DNA; D011088:DNA Ligases