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Direct detection and discrimination of nucleotide polymorphisms using anthraquinone labeled DNA probes

Direct detection and discrimination of nucleotide polymorphisms using anthraquinone labeled DNA probes
Direct detection and discrimination of nucleotide polymorphisms using anthraquinone labeled DNA probes
A novel electrochemical detection approach using DNA probes labeled with Anthraquinone (AQ) as a reporter moiety has been successfully exploited as a method for the direct detection of DNA targets. This assay uses simple voltammetry techniques (Differential Pulse Voltammetry) to exploit the unique responsiveness of AQ to its chemical environments within oxygenated aqueous buffers, providing a specific detection mechanism as a result of DNA hybridization. This measurement is based on a cathodic shift of the reduction potential of the AQ tag and the concurrent reduction in peak current upon DNA binding. The further utility of this approach for discrimination of closely related DNA targets is demonstrated using DNA strands specific to B. anthracis and closely related bacillus species. DNA targets were designed to the rpoB gene incorporating nucleotide polymorphisms associated with different bacillus species. This assay was used to demonstrate that the shift in reduction potential is directly related to the homology of the target DNA. The discriminatory mechanism is dependent on the presence of oxygen in the measurement buffer and is strongly linked to the position of the nucleotide polymorphisms; with homology at the terminus carrying the AQ functionalised nucleotide critical to achieving accurate discrimination. This understanding of assay design was used to demonstrate an optimized assay capable of discriminating between Yersinia pestis (the causative agent of plague) and closely related species based on the groEL gene. This method is attractive as it can not only detect DNA binding, but can also discriminate between multiple Single Nucleotide Polymorphisms (SNPs) within that DNA without the need for any additional reagents, reporters, or processes such as melting of DNA strands. This indicates that this approach may have great potential to be exploited within novel biosensors for detection and diagnosis of infectious disease in future Point of Care (PoC) devices.
2296-2646
Goodchild, Sarah A.
08211454-37d2-43e6-a41f-86efe8497adc
Gao, Rachel
47cbe2e0-7030-4348-a6df-35673fa2a253
Shenton, Daniel P.
8e3573a7-fcd4-4c95-b616-d85fd8ccb657
McIntosh, Alastair J.S.
385152df-376e-4d5d-b443-f3f01370b1c1
Brown, Tom
a64aae36-bb30-42df-88a2-11be394e8c89
Bartlett, Philip N.
d99446db-a59d-4f89-96eb-f64b5d8bb075
Goodchild, Sarah A.
08211454-37d2-43e6-a41f-86efe8497adc
Gao, Rachel
47cbe2e0-7030-4348-a6df-35673fa2a253
Shenton, Daniel P.
8e3573a7-fcd4-4c95-b616-d85fd8ccb657
McIntosh, Alastair J.S.
385152df-376e-4d5d-b443-f3f01370b1c1
Brown, Tom
a64aae36-bb30-42df-88a2-11be394e8c89
Bartlett, Philip N.
d99446db-a59d-4f89-96eb-f64b5d8bb075

Goodchild, Sarah A., Gao, Rachel, Shenton, Daniel P., McIntosh, Alastair J.S., Brown, Tom and Bartlett, Philip N. (2020) Direct detection and discrimination of nucleotide polymorphisms using anthraquinone labeled DNA probes. Frontiers in Chemistry. (doi:10.3389/fchem.2020.00381).

Record type: Article

Abstract

A novel electrochemical detection approach using DNA probes labeled with Anthraquinone (AQ) as a reporter moiety has been successfully exploited as a method for the direct detection of DNA targets. This assay uses simple voltammetry techniques (Differential Pulse Voltammetry) to exploit the unique responsiveness of AQ to its chemical environments within oxygenated aqueous buffers, providing a specific detection mechanism as a result of DNA hybridization. This measurement is based on a cathodic shift of the reduction potential of the AQ tag and the concurrent reduction in peak current upon DNA binding. The further utility of this approach for discrimination of closely related DNA targets is demonstrated using DNA strands specific to B. anthracis and closely related bacillus species. DNA targets were designed to the rpoB gene incorporating nucleotide polymorphisms associated with different bacillus species. This assay was used to demonstrate that the shift in reduction potential is directly related to the homology of the target DNA. The discriminatory mechanism is dependent on the presence of oxygen in the measurement buffer and is strongly linked to the position of the nucleotide polymorphisms; with homology at the terminus carrying the AQ functionalised nucleotide critical to achieving accurate discrimination. This understanding of assay design was used to demonstrate an optimized assay capable of discriminating between Yersinia pestis (the causative agent of plague) and closely related species based on the groEL gene. This method is attractive as it can not only detect DNA binding, but can also discriminate between multiple Single Nucleotide Polymorphisms (SNPs) within that DNA without the need for any additional reagents, reporters, or processes such as melting of DNA strands. This indicates that this approach may have great potential to be exploited within novel biosensors for detection and diagnosis of infectious disease in future Point of Care (PoC) devices.

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Accepted/In Press date: 14 April 2020
Published date: 12 May 2020

Identifiers

Local EPrints ID: 441833
URI: http://eprints.soton.ac.uk/id/eprint/441833
ISSN: 2296-2646
PURE UUID: 5f88ecb6-1fb3-4621-b974-4d5e2645ed7a
ORCID for Philip N. Bartlett: ORCID iD orcid.org/0000-0002-7300-6900

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Date deposited: 29 Jun 2020 16:33
Last modified: 29 Jul 2020 01:31

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Contributors

Author: Sarah A. Goodchild
Author: Rachel Gao
Author: Daniel P. Shenton
Author: Alastair J.S. McIntosh
Author: Tom Brown

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