Disposable sensors for rapid screening of mutated genes

• Published in: Analytical and bioanalytical chemistry 2010-10, Vol.398 (3), p.1385-1393
• Main Authors: García, T., Fernández-Barrena, M. G., Revenga-Parra, M., Núñez, A., Casero, E., Pariente, F., Prieto, J., Lorenzo, E.
• Format: Article
• Language: English
• Subjects: Analysis; Analytical Chemistry; Base Sequence; Biochemistry; Biosensing Techniques; Biosensors; Blood cells; Characterization and Evaluation of Materials; Chemical properties; Chemistry; Chemistry and Materials Science; Deoxyribonucleic acid; Design and construction; DNA - genetics; DNA Probes; Electric properties; Electrochemistry; Electrodes; Exact sciences and technology; Food Science; Gene mutations; General, instrumentation; Genes; High-throughput screening (Biochemical assaying); Identification and classification; Laboratory Medicine; Mathematical analysis; Methods; Molecular diagnostic techniques; Molecular probes; Monitoring/Environmental Analysis; Mutation; Mutations; Nucleic Acid Hybridization; Original Paper; Polymerase Chain Reaction; Polymorphism, Single Nucleotide; Reproducibility; Ruthenium; Screening; Single nucleotide polymorphisms; Standard deviation
• ISSN: 1618-2642; 1618-2650
• DOI: 10.1007/s00216-010-4029-5

A screening method for rapid detection of gene mutations directly in polymerase chain reaction (PCR) of genomic DNA is described. The method involves the development of a disposable screen-printed gold electrode modified with a thiolated capture probe directly obtained from denaturated PCR genomic DNA, which recognizes (by hybridization) its fully complementary sequence (wild type), giving a signal, whereas no signal is obtained for single-mismatched target (mutant). The detection of the hybridization event is achieved by changes in the metal redox center electroactivity of the complex [Ru(NH 3 ) 5  L] 2+ , where L is [3-(2-phenanthren-9-yl-vinyl)-pyridine], at –0.200 V. This complex binds to double-stranded DNA in a very selective form. The method allows discrimination between the wild type and the mutant of gene MRP3 directly in large PCR amplicons extracted from blood cells, without the need to use either synthetic probes or labeled targets. The mutation involves the presence of a single-nucleotide polymorphism (SNP) at base 54 of a 145-base-pair sequence from exon 21 of gene MRP3. Since the presence of this SNP might lead to a variety of hereditary liver disorders, its identification in a rapid and easy form may provide novel therapeutic targets for the future. The screening method proposed has excellent signal reproducibility, with a relative standard deviation of 10%. In addition, with the method developed as little as 6.6 ng/μL PCR product can be detected.