Document Detail


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.
MedLine Citation:
PMID:  23463388     Owner:  NLM     Status:  Publisher    
Abstract/OtherAbstract:
We developed an inexpensive laser induced fluorescence (LIF) dual-channel detection system and applied it to a ligase detection reaction (LDR)/capillary gel electrophoresis (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 non-labeled 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:
Mariko Hamada; Koji Shimase; Keiichi Noda; Kazuhiko Tsukagoshi; Masahiko Hashimoto
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Publication Detail:
Type:  JOURNAL ARTICLE     Date:  2013-3-6
Journal Detail:
Title:  Electrophoresis     Volume:  -     ISSN:  1522-2683     ISO Abbreviation:  Electrophoresis     Publication Date:  2013 Mar 
Date Detail:
Created Date:  2013-3-6     Completed Date:  -     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  8204476     Medline TA:  Electrophoresis     Country:  -    
Other Details:
Languages:  ENG     Pagination:  -     Citation Subset:  -    
Copyright Information:
© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Affiliation:
Department of Chemical Engineering and Materials Science, Faculty of Science and Engineering, Doshisha University, Kyotanabe, Kyoto, 610-0321, Japan.
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