Voltammetric analysis of 5-(4-Azidophenyl)-2 '-deoxycytidine nucleoside and azidophenyl-labelled single- and double-stranded DNAs
Abstrakt
Voltammetric determination of a redox labeled nucleoside 5-(4-azidophenyl)-2'-deoxycytidine (dC(AZP)) and various polymerase-synthesized dC(AZP)-labeled DNAs in aqueous buffers is presented. Influence of: i) pH (2-12), ii) scan rates (0.02-10 V s(-1)), and iii) dC(AZP) concentration (0.02-10 mu mol l(-1)), on voltammograms of dC(AZP) were systematically studied for the first time using CV at a hanging mercury drop electrode.
Electrode potential-controlled adsorption driven process allowed sensitive determination of dC(AZP) at nanomolar concentrations using adsorptive stripping voltammetry. Transfer stripping voltammetry (TSV) was used for the detection of dC(AZP)-labeled DNA in femtomole quantities.
Precise sequence-specific incorporation of dC(AZP) into DNA by primer extension was used to demonstrate a perfect correlation between the number of incorporated AZP moieties and TSV responses. In addition, for the first time we used polymerase chain reaction to prepare an about 350-bp double-stranded DNA fragment globally modified with dC(AZP), and of terminal deoxynucleotidyl transferase tailing reaction to generate end-labeled single stranded oligonucleotides.
Effects of DNA structure on the AZP-modified DNA TSV responses are discussed.