DETECTION AND QUANTIFICATION OF THE A3243G MUTATION OF MITOCHONDRIAL-DNA BY LIGATION DETECTION REACTION

The A3243G mutation of mitochondrial DNA is associated to the MELAS sy ndrome and to transmitted forms of diabetes mellitus. This mutation ex ists in a heteroplasmic state and can be present at a minor and hardly detectable It vel. The aim was to design a method which could be appl ied to large series of samples and could provide rapid, sensitive and quantitative detection of this mutation in the wild-type mitochondrial DNA background. The ability of ligation detection reaction (LDR) to s atisfy these objectives was evaluated. Ligation detection reaction was performed on a model template composed of mixtures of various proport ions of plasmids bearing the wild-type or mutant mitochondrial DNA seq uence. Radiolabelled or fluorescent primers and the wild-type and muta nt LDR products were separated by electrophoresis on conventional dena turating gel or on an Applied Biosystem 373. The ratios of mutant/wild -type products were consistent with the initial ratios of the plasmids in the template. The sensitivity and accuracy of the fluorescence and isotopic detection methods were similar. The detection limit of mutan t DNA was 10% of total mitochondrial DNA. The percentage of mutant DNA in DNA samples extracted from leukocytes of 19 patients having the mu tation at different levels, was evaluated by fluorescent or isotopic L DR. (C) 1998 Academic Press.