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 syndrome and to transmitted forms of diabetes mellitus. This mutation exists in a heteroplasmic state and can be present at a minor and hardly detectable level. The aim was to design a method which could be applied 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 satisfy these objectives was evaluated. Ligation detection reaction was performed on a model template composed of mixtures of various proportions of plasmids bearing the wild-type or mutant mitochondrial DNA sequence. Radiolabelled or fluorescent primers and the wild-type and mutant LDR products were separated by electrophoresis on conventional denaturating 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 mutant DNA was 10% of total mitochondrial DNA. The percentage of mutant DNA in DNA samples extracted from leukocytes of 19 patients having the mutation at different levels, was evaluated by fluorescent or isotopic LDR.
Laboratoire commun de biologie moléculaire, Hôpital Saint Antoine, Paris, France.
M
M
Nigou
B
B
Parfait
E
E
Clauser
J L
JL
Olivier
D001483:Base Sequence; D004247:DNA / Q000097:blood; D017931:DNA Primers; D004272:DNA, Mitochondrial / Q000235:genetics*; D006801:Humans; D007962:Leukocytes; D017241:MELAS Syndrome / Q000235:genetics*; D008969:Molecular Sequence Data; D010957:Plasmids; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D019914:Taq Polymerase; D013698:Templates, Genetic
D017931:DNA Primers; D004272:DNA, Mitochondrial; D004247:DNA; D019914:Taq Polymerase