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. Issue 9 (18th April 2013)

• Record Type: Journal Article
• Authors: Hamada, Mariko; Shimase, Koji; Noda, Keiichi; Tsukagoshi, Kazuhiko; Hashimoto, Masahiko
• Is Part Of: Electrophoresis. Volume 34:Issue 9/10(2013:May)
• Page Start: 1415
• Page End: 1422
• Publication Date: 2013-04-18
• Subjects: Electrophoresis -- Periodicals; Electrophoresis -- Periodicals; 541.372
• DOI: 10.1002/elps.201200671
• Languages: English
• ISSNs: 0173-0835

Abstract

<abstract abstract-type="main"> <title> <x xml:space="preserve">Abstract</x> </title> <p>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‐<italic>ras</italic> oncogene that have a high diagnostic value for colorectal cancer. LDR products generated from a particular K‐<italic>ras</italic> 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.</p> </abstract>