Alternating current voltammetric determination of DNA damage.
The conditions for alternating current (a.c.) voltammetric DNA determinations have been investigated with respect to its use with alkaline filter elution techniques at low DNA concentrations. In inorganic electrolyte solutions three current peaks can be distinguished: peak I around -1.1 V caused by the reorientation or desorption of DNA segments; peak II around -1.2 V caused by the native DNA (nDNA) form; peak III caused by denatured DNA (dDNA) at -1.4 V. Sonication of nDNA increases the peak current, however not with dDNA. Both dDNA and nDNA give linear peak current increments with DNA increments, their regression lines cutting the concentration axis at the origin. In filter elution techniques organic bases are often used. Adding ethanolamine (EA) elution buffer decreases the peak amplitude of DNA. It turns out that an unknown substance, perhaps a protein or RNA, elutes from the filters and gives rise to a current peak at about -1.3 V. This substance can interfere with the dDNA by competing for electrode surface area, since it diffuses much faster than the large molecules of the DNA. Since however, dDNA has a higher affinity for the electrode surface, after enough time, usually few minutes, the dDNA increasingly displaces the substance and occupies the surface. The same is valid for other organic molecules and thus also for EA. It is therefore remarkable that the unknown substance can be altered by ultrasonication, so that it will no longer interfere with dDNA, in contrast to EA. EA, on the other hand, can be "titrated". When EA is present at short accumulation times it prevents dDNA adsorption. By adding dDNA, the EA can be scavanged and further addition will adsorb and thus increase peak current in proportion to the concentration of the DNA present. The conditions for voltammetric DNA determination have been investigated obeying the recognized interactions. Avoiding organic bases and using inorganic ones would simplify the determination procedure. The reproducibility of the procedure in the range of 50-60 ng DNA/ml has been found to be +/- 6%.
Krznarić
D
D
Center for Marine Research Zagreb, Ruder Bosković Institute, Yugoslavia.
Cosović
B
B
Stüber
J
J
Zahn
R K
RK
D000818:Animals; D004247:DNA / Q000032:analysis*; D004249:DNA Damage; D004563:Electrochemistry; D006863:Hydrogen-Ion Concentration; D008297:Male; D012616:Sea Cucumbers; D013050:Spectrometry, Fluorescence
D004247:DNA