The previously reported approach of orthogonal multipotential redox coding of all four DNA bases allowed only analysis of the relative nucleotide composition of short DNA stretches. Here, we present two methods for normalization of the electrochemical readout to facilitate the determination of the total nucleotide composition. The first method is based on the presence or absence of an internal standard of 7-deaza-2'-deoxyguanosine in a DNA primer. The exact composition of the DNA was elucidated upon two parallel analyses and the subtraction of the electrochemical signal intensities. The second approach took advantage of a 5'-viologen modified primer, with this fifth orthogonal redox label acting as a reference for signal normalization, thus allowing accurate electrochemical sequence analysis in a single read. Both approaches were tested using various sequences, and the voltammetric signals obtained were normalized using either the internal standard or the reference label and demonstrated to be in perfect agreement with the actual nucleotide composition, highlighting the potential for targeted DNA sequence analysis.

• MeSH
• DNA Primers MeSH
• DNA * chemistry   MeSH
• Nucleotides * chemistry   MeSH
• Oxidation-Reduction MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA Primers MeSH
• DNA * MeSH
• Nucleotides * MeSH

Three sets of 7-deazaadenine and cytosine nucleosides and nucleoside triphosphates bearing either unsubstituted ferrocene, octamethylferrocene and ferrocenecarboxamide linked through an alkyne tether to position 7 or 5, respectively, were designed and synthesized. The modified dNFcX TPs were good substrates for KOD XL DNA polymerase in primer extension and were used for enzymatic synthesis of redox-labelled DNA probes. Square-wave voltammetry showed that the octamethylferrocene oxidation potential was shifted to lower values, whilst the ferrocenecarboxamide was shifted to higher potentials, as compared to ferrocene. Tailed PEX products containing different ratios of Fc-labelled A (dAFc ) and FcPa-labelled C (dCFcPa ) were synthesized and hybridized with capture oligonucleotides immobilized on gold electrodes to study the electrochemistry of the redox-labelled DNA. Clearly distinguishable, fully orthogonal and ratiometric peaks were observed for the dAFc and dCFcPa bases in DNA, demonstrating their potential for use in redox coding of nucleobases and for the direct electrochemical measurement of the relative ratio of nucleobases in an unknown sequence of DNA.

• Keywords
• DNA, electrochemistry, ferrocenes, nucleobases, redox labelling,
• MeSH
• Staining and Labeling methods   MeSH
• Cytidine Triphosphate chemistry   MeSH
• DNA Probes chemical synthesis   chemistry   MeSH
• DNA-Directed DNA Polymerase metabolism   MeSH
• DNA chemistry   metabolism   MeSH
• Electrochemical Techniques MeSH
• Metallocenes chemistry   MeSH
• Nucleotides chemistry   MeSH
• Oxidation-Reduction MeSH
• Substrate Specificity MeSH
• Ferrous Compounds chemistry   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Cytidine Triphosphate MeSH
• DNA Probes MeSH
• DNA-Directed DNA Polymerase MeSH
• DNA MeSH
• ferrocene MeSH Browser
• Metallocenes MeSH
• Nucleotides MeSH
• Ferrous Compounds MeSH

2'-Deoxyadenosine triphosphate (dATP) derivatives bearing diverse substituents (Cl, NH2 , CH3 , vinyl, ethynyl, and phenyl) at position 2 were prepared and tested as substrates for DNA polymerases. The 2-phenyl-dATP was not a substrate for DNA polymerases, but the dATPs bearing smaller substituents were good substrates in primer-extension experiments, producing DNA substituted in the minor groove. The vinyl-modified DNA was applied in thiol-ene addition and the ethynyl-modified DNA was applied in a CuAAC click reaction to form DNA labelled with fluorescent dyes in the minor groove.

• Keywords
• DNA modification, DNA polymerase, bioconjugation, fluorescent labelling, nucleotides,
• MeSH
• Nucleic Acid Denaturation MeSH
• Deoxyadenine Nucleotides chemistry   metabolism   MeSH
• DNA-Directed DNA Polymerase metabolism   MeSH
• DNA chemistry   metabolism   MeSH
• Nucleic Acid Conformation MeSH
• Base Sequence MeSH
• Substrate Specificity MeSH
• Thermodynamics MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• 2'-deoxyadenosine triphosphate MeSH Browser
• Deoxyadenine Nucleotides MeSH
• DNA-Directed DNA Polymerase MeSH
• DNA MeSH