Methylene blue as an electrochemical indicator for DF508 cystic fibrosis mutation detection.
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.
Nasef
Hany
H
Nanobiotechnology & Bioanalysis Group, Department of Chemical Engineering, Universitat Rovira i Virgili, Avinguda Països Catalans, 26, 43007 Tarragona, Spain.
Beni
Valerio
V
O'Sullivan
Ciara K
CK
D001483:Base Sequence; D003550:Cystic Fibrosis / Q000145:classification / Q000175:diagnosis* / Q000235:genetics*; D004252:DNA Mutational Analysis; D004563:Electrochemistry; D006801:Humans; D008751:Methylene Blue; D008954:Models, Biological; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D015341:Nucleic Acid Probes / Q000737:chemistry / Q000235:genetics; D016133:Polymerase Chain Reaction
D015341:Nucleic Acid Probes; D008751:Methylene Blue