Here, we report the electrochemical detection of single-point mutations using solid-phase isothermal primer elongation with redox-labeled oligonucleotides. A single-base mutation associated with resistance to rifampicin, an antibiotic commonly used for the treatment of Mycobacterium tuberculosis, was used as a model system to demonstrate a proof-of-concept of the approach. Four 5'-thiolated primers, designed to be complementary with the same fragment of the target sequence and differing only in the last base, addressing the polymorphic site, were self-assembled via chemisorption on individual gold electrodes of an array. Following hybridization with single-stranded DNA, Klenow (exo-) DNA polymerase-mediated primer extension with ferrocene-labeled 2'-deoxyribonucleoside triphosphates (dNFcTPs) was only observed to proceed at the electrode where there was full complementarity between the surface-tethered probe and the target DNA being interrogated. We tested all four ferrocenylethynyl-linked dNTPs and optimized the ratio of labeled/natural nucleotides to achieve maximum sensitivity. Following a 20 min hybridization step, Klenow (exo-) DNA polymerase-mediated primer elongation at 37 °C for 5 min was optimal for the enzymatic incorporation of a ferrocene-labeled nucleotide, achieving unequivocal electrochemical detection of a single-point mutation in 14 samples of genomic DNA extracted from Mycobacterium tuberculosis strains. The approach is rapid, cost-effective, facile, and can be extended to multiplexed electrochemical single-point mutation genotyping.

Departament d'Enginyeria Química Universitat Rovira i Virgili Avinguda Països Catalans 26 43007 Tarragona Spain

Department of Organic Chemistry Faculty of Science Charles University Hlavova 8 12843 Prague 2 Czech Republic

Faculty of Infectious and Tropical Diseases London School of Hygiene and Tropical Medicine WC1E 7HT London U K

Global Health and Tropical Medicine GHTM Instituto de Higiene e Medicina Tropical IHMT Universidade Nova de Lisboa Rua da Junqueira 100 1349 008 Lisbon Portugal

Institució Catalana de Recerca i Estudis Avançats Passeig Lluis Companys 23 08010 Barcelona Spain

Institute of Organic Chemistry and Biochemistry Czech Academy of Sciences Flemingovo nam 2 16610 Prague 6 Czech Republic

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