Electrochemical Detection of Single-Nucleotide Polymorphism Associated with Rifampicin Resistance in Mycobacterium tuberculosis Using Solid-Phase Primer Elongation with Ferrocene-Linked Redox-Labeled Nucleotides

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 (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.

Ferrocene as a potential electrochemical reporting surrogate of abasic sites in DNA

“…76 The next imminent steps towards the development of an electrochemical monitoring device will include square-wave voltammetry measurements on surface-immobilized oligonucleotides to examine the redox behavior of Fc-sites in the presence of natural and modified nucleotides. [77][78][79] Experimental section…”

Generic Platform for the Multiplexed Targeted Electrochemical Detection of Osteoporosis-Associated Single Nucleotide Polymorphisms Using Recombinase Polymerase Solid-Phase Primer Elongation and Ferrocene-Modified Nucleoside Triphosphates

“…In first demonstrations of the proof of concept, single SNPs were identified in DNA extracted and purified from sputum samples containing Mycobacterium tuberculosis to detect an SNP associated with rifampicin resistance 64 and then in a fingerprick blood sample to identify an SNP linked with cardiomyopathy. 59 In the present paper, we have extended our generic platform for the simultaneous detection of the five SNPs identified in the GEFOS/GENOMOS study.…”

Purine Nucleosides with a Reactive (β‐Iodovinyl)sulfone or a (β‐Keto)sulfone Group at the C8 Position and Their Polymerase‐Catalyzed Incorporation into DNA

“…Moreover, 8‐amino‐ATP, 8‐azido‐ATP, and 8‐aza‐ATP all produced RNA chain termination of polyadenylation while no primer extension was observed with 8‐Br‐ATP and 8‐Cl‐ATP [27] . Major groove modified 7‐substituted‐7‐deazapurine nucleoside triphosphates are better substrates for DNA polymerases than natural dATP and dGTP as they stabilize DNA duplexes [28–31] …”

“…[27] Major groove modified 7-substituted-7-deazapurine nucleoside triphosphates are better substrates for DNA polymerases than natural dATP and dGTP as they stabilize DNA duplexes. [28][29][30][31] Bioconjugation of the pyrimidine nucleotides bearing reactive groups with an amino acid, peptide or protein have been studied. [32][33][34][35] The Hocek group has cross-linked DNA fragments containing reactive vinylsulfonamide groups to peptides and protein p53.…”