DNA quantification via traceable phosphorus measurement through microwave-assisted UV digestion-ion chromatography

Abstract

Accurate quantification of deoxyribonucleic acid (DNA) is critical for many analyses in molecular biology and genetic tests. We present a method in which the stoichiometrically existing phosphorus content in purified genomic DNA is quantitatively converted into orthophosphate ions by microwave assisted-UV digestion in the presence of microlitre quantities of dilute reagents (HCl, HNO₃, H₂O₂). The tandem use of microwave energy and ultraviolet photons for DNA digestion in pressurized quartz vessels enables a maximum reaction temperature of 240 °C resulting in efficient and fast mineralization of high molecular weight DNA within 30 minutes. Compared to hotplate digestion, the digestion time is reduced by a factor of 32. The MW-UV sample preparation approach coupled with the ion chromatographic measurement of phosphate using a high performance (HP) methodology provides an accurate quantitation of phosphorus mass fractions as low as 0.3 μg g⁻¹, corresponding to a DNA mass of 25 μg. The relative expanded uncertainties (% U) expressed at 95% confidence for these analyses range from 0.2 to 0.6%. Critically, the matrix of the calibrant solution is also matched with respect to the digested matrix anions (chloride, nitrate), without which significant bias in IC performance is observed. The phosphorus content of the calf thymus DNA was also measured using high-performance inductively coupled plasma optical emission spectroscopy (HP-ICP-OES), which provided independent data for comparison with the MW-UV digestion-IC based approach. Ion chromatography requires smaller volume of materials to perform the analysis and could be useful for characterizing primary calibration standards and certified reference materials with low uncertainties.