Normalized Multipotential Redox Coding of DNA Bases for Determination of Total Nucleotide Composition

Identificador:  imarina:9329034

Handle: https://hdl.handle.net/20.500.11797/imarina9329034

Autors:  Kodr, David; Ortiz, Mayreli; Sykorova, Veronika; Yenice, Cansu Pinar; Lesnikowski, Zbigniew J; O'Sullivan, Ciara K; Hocek, Michal

Resum:
The previously reported approach of orthogonal multipotentialredoxcoding of all four DNA bases allowed only analysis of the relativenucleotide composition of short DNA stretches. Here, we present twomethods for normalization of the electrochemical readout to facilitatethe determination of the total nucleotide composition. The first methodis based on the presence or absence of an internal standard of 7-deaza-2 & PRIME;-deoxyguanosinein a DNA primer. The exact composition of the DNA was elucidated upontwo parallel analyses and the subtraction of the electrochemical signalintensities. The second approach took advantage of a 5 & PRIME;-viologenmodified primer, with this fifth orthogonal redox label acting asa reference for signal normalization, thus allowing accurate electrochemicalsequence analysis in a single read. Both approaches were tested usingvarious sequences, and the voltammetric signals obtained were normalizedusing either the internal standard or the reference label and demonstratedto be in perfect agreement with the actual nucleotide composition,highlighting the potential for targeted DNA sequence analysis.