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

作者：Hamada Mariko; Shimase Koji; Noda Keiichi; Tsukagoshi Kazuhiko; Hashimoto Masahiko^*

来源：Electrophoresis, 2013, 34(9-10): 1415-1422.

DOI：10.1002/elps.201200671

• Capillary gel electrophoresis
• Laser-induced fluorescence
• Ligase detection reaction
• Point mutation
• Single nucleotide variation

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.

• 出版日期2013-5