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.

Departament d'Enginyeria Química Universitat Rovira i Virgili 26 Països Catalans 43007 Tarragona Spain

Department of Organic Chemistry Faculty of Science Charles University Hlavova 8 Prague 2 CZ 12843 Czech Republic

Institució Catalana de Recerca i Estudis Avançats Passeig Lluís Companys 23 08010 Barcelona Spain

Institute of Organic Chemistry and Biochemistry Czech Academy of Sciences Flemingovo namesti 2 CZ 16000 Prague 6 Czech Republic

Laboratory of Medicinal Chemistry Institute of Medical Biology PAS Lodowa 106 92 232 Łódź Poland

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