Single-base mismatch detection based on charge transduction through DNA

Abstract

High-throughput DNA sensors capable of detecting single-base mismatches are required for the routine screening of genetic mutations and disease. A new strategy for the electrochemical detection of single-base mismatches in DNA has been developed based upon charge transport through DNA films. Double-helical DNA films on gold surfaces have been prepared and used to detect DNA mismatches electrochemically. The signals obtained from redox-active intercalators bound to DNA-modified gold surfaces display a marked sensitivity to the presence of base mismatches within the immobilized duplexes. Differential mismatch detection was accomplished irrespective of DNA sequence composition and mismatch identity. Single-base changes in sequences hybridized at the electrode surface are also detected accurately. Coupling the redox reactions of intercalated species to electrocatalytic processes in solution considerably increases the sensitivity of this assay. Reporting on the electronic structure of DNA, as opposed to the hybridization energetics of single-stranded oligonucleotides, electrochemical sensors based on charge transport may offer fundamental advantages in both scope and sensitivity.

Carbon and gold electrodes as electrochemical transducers for DNA hybridisation sensors

“…On the contrary, the detection scheme proposed by Barton and co-workers required the immobilisation of duplex monolayers so densely-packed that [Fe(CN) 6 ] 4− was electrochemically silent (Kelley et al, 1999a).…”

“…Both native and mutant test strands were allowed to associate with the capture probe under strongly hybridising conditions. Mismatch discrimination was then accomplished not by relying on the different thermodynamic stability of the duplexes, but measuring a change in electronic coupling within the base-pair stack (Kelley et al, 1999a;Boon et al, 2000).…”

“…A new strategy for the electrochemical detection of singlebase mismatches, based on DNA-mediated electron transfer, was developed by Barton's group (Kelley et al, 1999a). The presence of mismatches within the immobilised duplexes, caused a striking decrease in the electrochemical response of different redox-active intercalators (daunomycin, MB and [Ir(bpy)(phen)(phi)] 3+ ), non-covalently bound to the top of densely-packed DNA-modified gold surfaces.…”

DNA repair glycosylases with a [4Fe–4S] cluster: A redox cofactor for DNA-mediated charge transport?

“…Many tools have been developed to study DNA CT, including DNA-modified electrode systems [52][53]. In these systems, modified DNA duplexes are self-assembled into a monolayer via a thiol terminated tether on gold or through a pyrene-terminated linker on highly oriented pyrolytic graphite (HOPG) electrode surfaces.…”

Intercalative Stacking: A Critical Feature of DNA Charge-Transport Electrochemistry

“…[52][53][54][55][56][57][58][59] The electrochemistry, binding affinity, and charge-transport dynamics of intercalated organic dyes at these electrodes have been examined. 52,54,55 Noncovalent binding appears to be primarily constrained to the top of the densely packed DNA monolayer, as established previously with daunomycin 55 and methylene blue. 52 Electrochemical studies involving daunomycin site-specifically bound at various positions within DNA monolayers reveal that DNA-mediated charge transport proceeds over significant distances (>35 Å).…”