Experimental design applied to the optimization of microwave-assisted DNA hydrolysis

The assessment of the integrity of the DNA primary structure and the identification of canonical and modified bases are useful tools in medical, pharmaceutical, and forensic applications. In this article we report on the first microwave-assisted hydrolyses of deoxyribonucleoside-triphosphates (dNTPs) and human DNA using "Design of Experiments" methodology. We use hydrophilic interaction chromatography (HILIC) and UV detection at 260nm for the determination of purinic and pyrimidinic bases at levels of 0.5μM. We use a ZIC-HILIC ® 150mm×2.1mm i.d., 5μm particle size column and ammonium formate buffers in acetonitrile for gradient separation of the analytes. We then compare the final concentrations of Thymine, Adenine, Cytosine, and Guanine with the nominal amounts of such bases in the dNTPs and DNA submitted to hydrolysis. After optimization of the hydrolysis (11.5min, 0.15M aqueous HCl, 150°C), the method turns out to be suitable for the determination of products released from quantities of human DNA as low as 500ng with precision (RSD<10%) and accuracy (REC 97-104%). These results confirm that the kinetics of the release of the bases depends on their molecular structure and show that the concentration of the substrate plays a relevant role. We conclude with a discussion of the method and a comparison to the methods described in previous studies