Cystic fibrosis is one of the most common life-shortening, childhood-onset inherited diseases. Among the 1,000 known cystic fibrosis-related mutations, DF508 is the most common, with a frequency varying between 50% and 70% according to geographical areas and population typology. In this work, we report the use of methylene blue as an electrochemical reporting agent in the discrimination of synthetic PCR analogue of the DF508 cystic fibrosis mutation (Mut) from the wild type (Wt). At optimum experimental condition, a discrimination factor between mutant and wild type of approximately 1.5-fold was found. The proposed assay was quantitative and linear in the range of 10-100 nM, exhibiting a limit of detection of 2.64 nM. Electrochemical studies at variable ionic strength conditions allowed further elucidation of the mechanism of the methylene blue (MB)-DNA interaction. To the best of our knowledge, this is the first report of detection of hybridisation solely via guanine-specific MB-DNA interaction simultaneously in MB solution, independent of electrostatic interaction as demonstrated in the ionic strength study. The introduction of formamide in the hybridization buffer, to improve discrimination, was also investigated. Finally, mutant wild type discrimination was demonstrated, at 10 nM concentration, with the use of a multi-sensor setup.
Cystic fibrosis is one of the most common genetically inherited diseases in northern Europe, with the DF508 mutation being the most common, and among the Caucasian population being responsible for almost 70% of cases. In this work, we report on the use of thermally modulated electrochemical impedance spectroscopy for the discrimination of the DF508 mutation from the wild-type sequence. DNA probes (15 and 21 bases long) were immobilised on the surface of gold electrodes and the variation of the charge-transfer resistance was monitored as a function of hybridisation. Two sets of targets were used in this work: synthetic 15-mer sequences and two single-stranded synthetic analogues of PCR products 82 (mutant) and 85 (wild type) bases long. Hybridisation with short targets resulted in very sequence specific charge-transfer-resistance variation with a discrimination factor at room temperature between fully complementary and mismatched sequences of approximately fivefold. However, in the case of the single-stranded synthetic PCR product analogues, a lower discrimination factor was recorded (1.5-fold). The effect of temperature was investigated to improve discrimination and the use of a posthybridisation wash at elevated temperature resulted in a fivefold improvement in the discrimination factor. Using an electrode array with probes immobilised against each of the mutant and wild-type sequences, we achieved an unequivocal detection of the DF508 mutation.
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