title: Microbead-based ligase detection reaction assay using a molecular beacon probe for the detection of low-abundance point mutations.
abstract: A microbead-based ligase detection reaction (LDR) assay using a molecular beacon probe was developed for the facile and rapid detection of point mutations present in low copy numbers in a mixed population of wild-type DNA. Biotin-tagged ligation products generated in the LDR were captured on the surface of streptavidin-modified magnetic beads for purification and concentration. The resulting product-tethered microbeads were combined with a molecular beacon probe solution, and the suspension was directly flowed into a capillary. The microbeads were accumulated in a confined space within the capillary using a bar magnet. The packed bead sample was then scanned by a fluorescence scanning imager to detect the presence of any mutations. With the developed methodology, we were able to successfully detect one cancer mutation in a mixture of 400 wild-type templates (t test at 95% confidence level). Furthermore, the post-LDR processing, typically the most laborious and time-consuming step in LDR-based mutation detection assays, could be carried out much more rapidly (approximately 20 min). This was enabled by the simple bead and fluid manipulations involved in the present assay.
authors: Department of Chemical Engineering and Materials Science, Faculty of Science and Engineering, Doshisha University , Kyotanabe, Kyoto 610-0321, Japan.
authors: Sho
authors: S
authors: Watanabe
authors: Kenta
authors: K
authors: Hagihara
authors: Kazuhiko
authors: K
authors: Tsukagoshi
authors: Masahiko
authors: M
authors: Hashimoto
mesh_terms: D000595:Amino Acid Sequence; D006801:Humans; D008025:Ligases / Q000032:analysis* / Q000235:genetics*; D008863:Microspheres; D015335:Molecular Probes / Q000737:chemistry* / Q000235:genetics*; D008969:Molecular Sequence Data; D017354:Point Mutation / Q000235:genetics*
chemical_list: D015335:Molecular Probes; D008025:Ligases