title: Development of a PCR/LDR/flow-through hybridization assay using a capillary tube, probe DNA-immobilized magnetic beads and chemiluminescence detection.
abstract: A polymerase chain reaction (PCR)/ligase detection reaction (LDR)/flow-through hybridization assay using chemiluminescence (CL) detection was developed for analyzing point mutations in gene fragments with high diagnostic value for colorectal cancers. A flow-through hybridization format using a capillary tube, in which probe DNA-immobilized magnetic beads were packed, provided accelerated hybridization kinetics of target DNA (i.e. LDR product) to the probe DNA. Simple fluid manipulations enabled both allele-specific hybridization and the removal of non-specifically bound DNA in the wash step. Furthermore, the use of CL detection greatly simplified the detection scheme, since CL does not require a light source for excitation of the fluorescent dye tags on the LDR products. Preliminary results demonstrated that this analytical system could detect both homozygous and heterozygous mutations, without the expensive instrumentation and cumbersome procedures required by conventional DNA microarray-based methods.
authors.affiliation: Department of Chemical Engineering and Materials Science, Faculty of Science and Engineering, Doshisha University, Kyotanabe, Kyoto, Japan.
authors.forename: Manami
authors.initials: M
authors.lastname: Hommatsu
authors.forename: Hisamitsu
authors.initials: H
authors.lastname: Okahashi
authors.forename: Keisuke
authors.initials: K
authors.lastname: Ohta
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Tamai
authors.forename: Kazuhiko
authors.initials: K
authors.lastname: Tsukagoshi
authors.forename: Masahiko
authors.initials: M
authors.lastname: Hashimoto
mesh_terms: D015342:DNA Probes; D011905:Genes, ras; D049449:Luminescence; D008280:Magnetics; D009154:Mutation; D009693:Nucleic Acid Hybridization / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods*
chemical_list: D015342:DNA Probes