Autor según el artículo: Ortiz M; Jauset-Rubio M; Skouridou V; MacHado D; Viveiros M; Clark TG; Simonova A; Kodr D; Hocek M; O'Sullivan CK
Departamento: Enginyeria Química
Autor/es de la URV: Jauset Rubio, Miriam / O'SULLIVAN, CIARA KATHLEEN / Ortíz Rodríguez, Mayreli / Skouridou, Vasoula
Palabras clave: Solid-phase primer elongation Solid-phase Solid phasis Single-point mutation Single-nucleotide polymorphism (snp) Single-nucleotide polymorphism Single nucleotide polymorphisms Polymorphism Organometallics Oligonucleotides Nucleoside triphosphates Klenow (exo-) dna polymerase Iron compounds Ferrocene-labeled nucleotides Ferrocene-labeled nucleotide Ferrocene labelled Elongation Electrodes Dna polymerase Dna Cost effectiveness Chemical detection
Resumen: 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.
Áreas temáticas: Process chemistry and technology Nanoscience & nanotechnology Instrumentation Fluid flow and transfer processes Engenharias iii Chemistry, multidisciplinary Chemistry, analytical Bioengineering
Acceso a la licencia de uso: https://creativecommons.org/licenses/by/3.0/es/
Direcció de correo del autor: miriam.jauset@urv.cat vasoula.skouridou@urv.cat mayreli.ortiz@urv.cat
Identificador del autor: 0000-0002-9943-6132 0000-0002-9712-5429 0000-0002-9423-0055
Fecha de alta del registro: 2023-02-19
Versión del articulo depositado: info:eu-repo/semantics/publishedVersion
Enlace a la fuente original: https://pubs.acs.org/doi/10.1021/acssensors.1c01710
URL Documento de licencia: http://repositori.urv.cat/ca/proteccio-de-dades/
Referencia al articulo segun fuente origial: Acs Sensors. 6 (12): 4398-4407
Referencia de l'ítem segons les normes APA: Ortiz M; Jauset-Rubio M; Skouridou V; MacHado D; Viveiros M; Clark TG; Simonova A; Kodr D; Hocek M; O'Sullivan CK (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
DOI del artículo: 10.1021/acssensors.1c01710
Entidad: Universitat Rovira i Virgili
Año de publicación de la revista: 2021
Tipo de publicación: Journal Publications
Repositori URV