title: Experimental design applied to the optimization of microwave-assisted DNA hydrolysis.
abstract: 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 260 nm for the determination of purinic and pyrimidinic bases at levels of 0.5 μM. We use a ZIC-HILIC 150mm × 2.1 mm i.d., 5 μm 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 (11.5 min, 0.15 M 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 500 ng 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.
authors: Department of Drug Sciences, University of Pavia, viale Taramelli 12, 27100 Pavia, Italy. giorgio.marrubini@unipv.it
authors: Giorgio
authors: G
authors: Marrubini
authors: Paolo
authors: P
authors: Fattorini
authors: Carlo
authors: C
authors: Previderé
authors: Silvia
authors: S
authors: Goi
authors: Solange
authors: S
authors: Sorçaburu Cigliero
authors: Pierangela
authors: P
authors: Grignani
authors: Massimo
authors: M
authors: Serra
authors: Raffaela
authors: R
authors: Biesuz
authors: Gabriella
authors: G
authors: Massolini
mesh_terms: D002138:Calibration; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000737:chemistry*; D006868:Hydrolysis; D008872:Microwaves; D012015:Reference Standards; D013056:Spectrophotometry, Ultraviolet
chemical_list: D004247:DNA