| Autor: |
Ortiz M; Departament d'Enginyeria Química, Universitat Rovira i Virgili, Avinguda Països Catalans 26, 43007 Tarragona, Spain., Jauset-Rubio M; Departament d'Enginyeria Química, Universitat Rovira i Virgili, Avinguda Països Catalans 26, 43007 Tarragona, Spain., Skouridou V; Departament d'Enginyeria Química, Universitat Rovira i Virgili, Avinguda Països Catalans 26, 43007 Tarragona, Spain., Machado D; 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., Viveiros M; 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., Clark TG; 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.; Faculty of Infectious and Tropical Diseases, London School of Hygiene & Tropical Medicine, WC1E 7HT London, U.K., Simonova A; Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Flemingovo nam.2, 16610 Prague 6, Czech Republic.; Department of Organic Chemistry, Faculty of Science, Charles University, Hlavova 8, 12843 Prague 2, Czech Republic., Kodr D; Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Flemingovo nam.2, 16610 Prague 6, Czech Republic., Hocek M; Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Flemingovo nam.2, 16610 Prague 6, Czech Republic.; Department of Organic Chemistry, Faculty of Science, Charles University, Hlavova 8, 12843 Prague 2, Czech Republic., O'Sullivan CK; Departament d'Enginyeria Química, Universitat Rovira i Virgili, Avinguda Països Catalans 26, 43007 Tarragona, Spain.; Institució Catalana de Recerca i Estudis Avançats, Passeig Lluis Companys 23, 08010 Barcelona, Spain. |
| Abstrakt: |
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 (dN Fc TPs) 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. |
