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.

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  • Author(s): Hamada M;Hamada M; Shimase K; Noda K; Tsukagoshi K; Hashimoto M
  • Source:
    Electrophoresis [Electrophoresis] 2013 May; Vol. 34 (9-10), pp. 1415-22. Date of Electronic Publication: 2013 Apr 18.
  • Publication Type:
    Journal Article; Research Support, Non-U.S. Gov't
  • Language:
    English
  • Additional Information
    • Source:
      Publisher: Wiley-VCH Country of Publication: Germany NLM ID: 8204476 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1522-2683 (Electronic) Linking ISSN: 01730835 NLM ISO Abbreviation: Electrophoresis Subsets: MEDLINE
    • Publication Information:
      Publication: : Weinheim : Wiley-VCH
      Original Publication: [Weinheim, Germany] : Verlag Chemie, [1980-
    • Subject Terms:
      Genes, ras* ; Point Mutation*; DNA/*genetics ; Electrophoresis, Capillary/*instrumentation; Alleles ; DNA/metabolism ; DNA Ligases/metabolism ; Electrophoresis, Capillary/methods ; Equipment Design ; Fluorescence ; Fluorescent Dyes/analysis ; Lasers
    • 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.
      (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)
    • Accession Number:
      0 (Fluorescent Dyes)
      9007-49-2 (DNA)
      EC 6.5.1.- (DNA Ligases)
    • Publication Date:
      Date Created: 20130307 Date Completed: 20131022 Latest Revision: 20130514
    • Publication Date:
      20231215
    • Accession Number:
      10.1002/elps.201200671
    • Accession Number:
      23463388