Repositori institucional URV
| URV's Author/s: | Jauset Rubio, Miriam / O'SULLIVAN, CIARA KATHLEEN / Ortíz Rodríguez, Mayreli / Skouridou, Vasoula |
| Author, as appears in the article.: | Ortiz, Mayreli; Jauset-Rubio, Miriam; Skouridou, Vasso; Machado, Diana; Viveiros, Miguel; Clark, Taane G; Simonova, Anna; Kodr, David; Hocek, Michal; O'Sullivan, Ciara K |
| Author's mail: | vasoula.skouridou@urv.cat mayreli.ortiz@urv.cat miriam.jauset@urv.cat mayreli.ortiz@urv.cat |
| Author identifier: | 0000-0002-9712-5429 0000-0002-9423-0055 0000-0002-9943-6132 0000-0002-9423-0055 |
| Journal publication year: | 2021 |
| Publication Type: | Journal Publications |
| APA: | Ortiz, Mayreli; Jauset-Rubio, Miriam; Skouridou, Vasso; Machado, Diana; Viveiros, Miguel; Clark, Taane G; Simonova, Anna; Kodr, David; Hocek, Michal; (2021). Electrochemical Detection of Single-Nucleotide Polymorphism Associated with Rifampicin Resistance in Mycobacterium tuberculosis Using Solid-Phase Primer Elongation with Ferrocene-Linked Redox-Labeled Nucleotides. Acs Sensors, 6(12), 4398-4407. DOI: 10.1021/acssensors.1c01710 |
| Paper original source: | Acs Sensors. 6 (12): 4398-4407 |
| Abstract: | 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. © 2021 The Authors. Published by American Chemical Society. |
| Article's DOI: | 10.1021/acssensors.1c01710 |
| Link to the original source: | https://pubs.acs.org/doi/10.1021/acssensors.1c01710 |
| Paper version: | info:eu-repo/semantics/publishedVersion |
| licence for use: | https://creativecommons.org/licenses/by/3.0/es/ |
| Department: | Enginyeria Química |
| Licence document URL: | https://repositori.urv.cat/ca/proteccio-de-dades/ |
| Thematic Areas: | Process chemistry and technology Nanoscience & nanotechnology Instrumentation Fluid flow and transfer processes Engenharias iii Chemistry, multidisciplinary Chemistry, analytical Bioengineering |
| Keywords: | Solid-phase primer elongation Solid-phase Solid phasis Single-point mutation Single-nucleotide polymorphism (snp) Single-nucleotide polymorphism Single nucleotide polymorphisms Rifampin Polymorphism, single nucleotide Polymorphism Oxidation-reduction Organometallics Oligonucleotides Nucleoside triphosphates Mycobacterium tuberculosis Metallocenes Klenow (exo-) dna polymerase Iron compounds Ferrocene-labeled nucleotides Ferrocene-labeled nucleotide Ferrocene labelled Elongation Electrodes Dna polymerase Dna Cost effectiveness Chemical detection |
| Entity: | Universitat Rovira i Virgili |
| Record's date: | 2024-10-12 |
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