Voltammetric determination of all DNA nucleotides
Section snippets
Materials and reagents
Guanine, adenine, thymine, cytosine, guanosine 5-monophosphate (GMP), adenosine 5-monophosphate (AMP), thymidine 5-monophosphate (TMP), cytidine 5-monophosphate (CMP), lyophilized calf thymus single-stranded DNA, sodium salt poly(dT), and sodium salt poly(dC) were obtained from Sigma–Aldrich and used without further purification. Analytical-grade reagents and purified water from a Millipore Milli-Q system (conductivity <0.1 μS cm−1) were used for the preparation of phosphate, acetate, borate, and
Results and discussion
In previous work [14], [15], [16] the oxidation of all DNA bases was detected but in solutions containing pyrimidine bases with a concentration 10 times higher than the purine base concentration.
The differential pulse voltammogram obtained for a mixture of 20 μM guanine and adenine and 200 μM thymine and cytosine in pH 7.4, 0.1 M phosphate buffer supporting electrolyte solution using a 3-mm-diameter GCE shows four oxidation peaks of the same height magnitude (Fig. 1). The peak at 0.70 V is
Conclusion
The study of DNA oxidative damage is of great importance since it is well established that it constitutes an important source of genomic instability. Voltammetric methods are suitable to study DNA oxidation, but almost all previous studies focus only on the oxidation of purine derivative compounds.
The voltammetric results presented in this work extend previous voltammetric studies on DNA to include all four bases and corresponding nucleotides. For the first time equimolar mixtures of all DNA
Acknowledgments
Financial support from Fundae¸ ão para a Ciência e Tecnologia (FCT), Ph.D. Grants PRAXIS XXI/BD/6134/2001 (J.A.P.P.) and PRAXIS SFRH/BD/877/2000 (V.C.D.), projects PRAXIS/P/AGR/12205/1998, POCTI (cofinanced by the European Community Fund FEDER), ICEMS (Research Unit 103), and European Projects QLK3-2000-01311 and HPRN-CT-2002-00186 are gratefully acknowledged.
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