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
Department: Enginyeria Química
URV's Author/s: Jauset Rubio, Miriam / O'SULLIVAN, CIARA KATHLEEN / Ortíz Rodríguez, Mayreli / Skouridou, Vasoula
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
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.
Thematic Areas: Process chemistry and technology Nanoscience & nanotechnology Instrumentation Fluid flow and transfer processes Engenharias iii Chemistry, multidisciplinary Chemistry, analytical Bioengineering
licence for use: https://creativecommons.org/licenses/by/3.0/es/
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
Record's date: 2024-10-12
Papper version: info:eu-repo/semantics/publishedVersion
Link to the original source: https://pubs.acs.org/doi/10.1021/acssensors.1c01710
Licence document URL: https://repositori.urv.cat/ca/proteccio-de-dades/
Papper original source: Acs Sensors. 6 (12): 4398-4407
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
Article's DOI: 10.1021/acssensors.1c01710
Entity: Universitat Rovira i Virgili
Journal publication year: 2021
Publication Type: Journal Publications